Regulators of NFAT

ABSTRACT

Disclosed are methods of identifying an agent that modulates an NFAT regulator protein. One such method comprises contacting at least one test agent with a recombinant cell comprising at least one NFAT regulator protein or fragment or derivative thereof, assessing the effect of the test agent on an activity, interaction, expression, or binding to the NFAT regulator protein or fragment or derivative thereof, and identifying the test agent that has an effect on an activity, interaction, expression, or binding to the NFAT regulator protein or fragment or derivative thereof, whereby the identified test agent is characterized as an agent that modulates an NFAT regulator protein. Methods of identifying an agent that modulates intracellular calcium, methods to screen for an agent that modulates NFAT regulator function, methods to diagnose unexplained immunodeficiency in a subject, and methods for identifying an agent for treating or preventing a disease or disorder associated with a NFAT regulator protein or calcium signaling are also disclosed.

RELATED APPLICATIONS

This Application claims priority to U.S. Provisional Application 60/756,934, filed, Jan. 5, 2006, the entire contents of which are incorporated by reference herein.

GOVERNMENT SUPPORT

This invention was supported, in part, by National Institutes of Health (NIH) Grant Nos. RO1 A140127, HD39685, R21AI054933, and GM 075256. The government of the United States has certain rights to the invention.

FIELD OF THE INVENTION

The invention relates to the field of regulation of a family of calcium regulated transcription factors known as NFAT proteins.

BACKGROUND OF THE INVENTION

Hyperactivity or inappropriate activity of the immune system is a serious and widespread medical problem. It contributes to acute and chronic immune diseases, e.g., allergic and atopic diseases, e.g., asthma, allergic rhinitis, allergic conjunctivitis and atopic dermatitis, and to autoimmune diseases, e.g., rheumatoid arthritis, insulin-dependent diabetes, inflammatory bowel disease, autoimmune thyroiditis, hemolytic anemia and multiple sclerosis. Hyperactivity or inappropriate activity of the immune system is also involved in transplant graft rejections and graft-versus-host disease.

A certain family of transcription factors, the NFAT proteins (nuclear factor of activated T cells), are expressed in immune cells and play a key role in eliciting immune responses. The NFAT proteins are activated by an increase in intracellular calcium levels, e.g., by means of store-operated calcium entry. The activated NFAT proteins, in turn, induce transcription of cytokine genes which are required for an immune response. The immunosuppressive drugs cyclosporin A and FK506 are potent inhibitors of cytokine gene transcription in activated immune cells, and have been reported to act by inhibiting calcineurin such that calcineurin is not able to activate NFAT. These drugs, however, can display nephrotoxic and neurotoxic effects after long term usage. Since calcineurin is ubiquitously expressed in many tissues, the drugs' inhibition of calcineurin activity toward substrates other than NFAT may contribute to the observed toxicity.

There is a need for immunosuppressive agents which selectively inhibit the store-operated calcium entry activation of NFAT.

SUMMARY OF THE INVENTION

The present invention provides a method for identifying an agent that modulates NFAT activity. In one embodiment, the agent modulates NFAT activity by means of modulating intracellular calcium levels. In one preferred embodiment, the agent modulates at least one component of the CRAC channel, e.g., an ORAI protein, e.g., proteins encoded by ORAI1 (NM_(—)032790; SEQ ID NO: 1), ORAI2 (BC069270; SEQ ID NO: 2), and/or ORAI3 (NM_(—)152288; SEQ ID NO: 3). In one embodiment, the agent modulates phosphorylation of NFAT, e.g., via modulation of a DYRK protein, e.g., proteins encoded by DYRK1A (NM_(—)001396; SEQ ID NO:4), DYRK1B (NM_(—)004714; SEQ ID NO:5), DYRK2 (NM_(—)003583; SEQ ID NO:6), DYRK3 (NM_(—)003582; SEQ ID NO:7), DYRK4 (NM_(—)003845; SEQ ID NO:8) and/or DYRK6 (NM_(—)005734; SEQ ID NO:9).

The present invention provides a method of identifying an agent that modulates an NFAT regulator protein, comprising contacting at least one test agent with a recombinant cell comprising at least one NFAT regulator protein or fragment or derivative thereof; assessing the effect of the test agent on an activity, interaction, expression, or binding to the NFAT regulator protein or fragment or derivative thereof; and identifying the test agent that has an effect on an activity, interaction, expression, or binding to the NFAT regulator protein or fragment or derivative thereof, whereby the identified test agent is characterized as an agent that modulates an NFAT regulator protein.

In one embodiment, the NFAT regulator protein is encoded by at least one NFAT regulator selected from the group consisting of ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO:2), ORAI3 (SEQ ID NO:3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) and DYRK6 (SEQ ID NO:9). In one embodiment, the NFAT regulator protein is encoded by at least one of the genes listed in Table I.

In one embodiment, assessing the effect of the test agent comprises using an antibody which specifically binds to a NFAT regulator protein encoded by ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8), or DYRK6 (SEQ ID NO:9).

In one embodiment, the method further comprises assessing the effect of the test agent on electrical current across the plasma membrane of the cell. In one embodiment, the electrical current is due to flux of monovalent cations or divalent cations across the cell. In one embodiment, the method further comprises assessing the effect of the test agent on intracellular calcium within the cell. In one embodiment, the method further comprises identifying the test agent that has an effect on intracellular calcium within the cell, whereby the identified test agent is characterized as an agent that modulates intracellular calcium and an agent that modulates NFAT regulator protein.

In one embodiment, the cell comprises at least one heterologous NFAT regulator proteins or a fragment or derivative thereof. In one embodiment, the cell comprises heterologous nucleic acid encoding at least one NFAT regulator protein or a fragment or derivative thereof. In one embodiment, the cell overexpresses, or underexpresses at least one NFAT regulator protein or fragment or derivative thereof.

The present invention further provides a method of identifying an agent that modulates

intracellular calcium, comprising contacting at least one test agent with a recombinant cell comprising at least one NFAT regulator protein or fragment or derivative thereof; assessing the effect(s) of the test agent on intracellular amounts, or concentrations, of cations or divalent cations within the cell, or on ion influx into the cell; and identifying the test agent that has an effect on intracellular amounts or concentrations of cations or divalent cations within the cell, or on ion influx into the cell, whereby the identified test agent is characterized as an agent that modulates intracellular calcium. In one embodiment, the intracellular cation is calcium. In one embodiment, assessing the effect of the test agent comprises monitoring calcium levels in the cytoplasm, monitoring calcium levels in an intracellular calcium store, monitoring calcium movement, or monitoring a calcium-entry mediated event. In one embodiment, the method further comprises assessing the effect of the test agent on an activity, interaction, expression, or binding to the NFAT regulator protein or fragment or derivative thereof. In one embodiment, the NFAT regulator protein is encoded by at least one NFAT regulator selected from the group consisting of ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) or DYRK6 (SEQ ID NO:9). In one embodiment, the agent that modulates intracellular calcium is further characterized as an agent that modulates NFAT regulator protein. In one embodiment, the recombinant cell comprises at least one heterologous NFAT regulator proteins or a fragment or derivative thereof. In one embodiment, the recombinant cell comprises a heterologous nucleic acid encoding at least one NFAT regulator proteins or fragment or derivative thereof. In one embodiment, the recombinant cell overexpresses at least one NFAT regulator protein or fragment or derivative thereof. In one embodiment, the recombinant cell exhibits dyshomeostasis. In one embodiment, the recombinant cell exhibits calcium dyshomeostasis

The present invention further provides a method to screen for an agent that modulates NFAT regulator function, comprising administering at least one test agent to a recombinant cell comprising at least one vector that comprises heterologous nucleic acid encoding at least one NFAT regulatory domain or a fragment or derivative thereof, operably linked to a sequence encoding a reporter protein; and monitoring intracellular localization of at least one expression product encoded by the vector, whereby a test agent that has an effect on intracellular localization of the expression product is characterized as an agent that modulates NFAT regulator function. In one embodiment, the agent that modulates NFAT regulator function is associated with cytoplasmic or nuclear localization of the expression product. In one embodiment, the cell is under resting conditions. In one embodiment, the cell is stimulated with a calcium modulating agent. In one embodiment, the cell is stimulated with thapsigargin or ionomycin. In one embodiment, the cell is further administered a vector that comprises a heterologous nucleic acid encoding at least one NFAT regulator protein, or a fragment or derivative thereof. In one embodiment, the vector that comprises the heterologous nucleic acid encoding at least one NFAT regulator protein, or fragment or derivative thereof, is the same vector that comprises heterologous nucleic acid encoding at least one NFAT regulatory domain or a fragment or derivative thereof, operably linked to a sequence encoding a reporter protein.

The present invention further provides a method to diagnose unexplained immunodeficiency in a subject comprising sequencing at least 25 contiguous nucleotides in a gene from the subject corresponding to ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO:2), ORAI3 (SEQ ID NO:3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID N07), DYRK4 (SEQ ID NO:8), DYRK6 (SEQ ID NO:9), or any of the genes listed in Table I; and comparing the sequence of the subject's gene to the wild type sequence of the gene, wherein a variation between the gene from the wild type sequence indicates the subject's gene is responsible for the immunodeficiency. In one embodiment, the comparison comprises obtaining a biological sample from the subject, sequencing the DNA in the biological sample, and electronically aligning the DNA sequence obtained from the biological sample to a wild type sequence. In one embodiment, the variation comprises a nucleotide mutation from C to T at position 271 of the coding sequence of ORAI1 (SEQ ID NO: 1). In one embodiment, the unexplained immunodeficiency is associated with defects in regulation of NFAT activity. In one embodiment, the variation comprises a mutation in a splice site. In one embodiment, the variation comprises a nonsynonymous mutation.

The present invention further provides a method for identifying an agent for treating or preventing a disease or disorder associated with a NFAT regulator protein, comprising assessing the effects of a test agent on an organism exhibiting a disease or disorder associated with NFAT regulator protein; and identifying the test agent as an agent for treating or preventing a disease or disorder associated with NFAT regulator protein if it has an effect on a phenotype of the organism associated with the disease or disorder, wherein the test agent modulates an activity, interaction, expression, or binding of, at least one NFAT regulator protein or fragment or derivative thereof. In one embodiment, the organism comprises one or more cells that exhibit calcium dyshomeostasis. In one embodiment, the organism exhibits calcium dyshomeostasis. In one embodiment, the phenotype on which the test agent has an effect is associated with the disease or disorder. This method is particularly useful, for diseases or conditions associated with altered regulation of intracellular calcium.

In one embodiment, the disease or disorder is primarily attributable to deranged calcium signaling. In one embodiment, the disease or disorder associated with NFAT regulator protein is rheumatoid arthritis, inflammatory bowel disease, allogeneic or xenogeneic transplantation rejection, graft-versus-host disease, aplastic anemia, psoriasis, lupus erytematosus, inflammatory disease, MS, type I diabetes, asthma, pulmonary fibrosis, scleroderma, dermatomyositis, Sjogren's syndrome, postpericardiotomy syndrome, Kawasaki disease, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pemphigus vulgaris, autoimmune hemolytic anemia, idiopathic thrombopenia, chronic glomerulonephritis, Goodpasture's syndrome, Wegner's granulomatosis, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, uveitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's disease, ulcerative colitis, colitis/inflammatory bowel syndrome, Guilllain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, eczema, and autoimmune thyroiditis. Transplant graft rejections, acquired immunodeficiencies, common variable immunodeficiency, myocardial hypertrophy, severe combined immunodeficiency, dilated cardiomyopathy, excessive or pathological bone resorption, excessive adipocyte differentiation, obesity, or reactivation of latent viruses.

The present invention further provides an antibody which specifically binds to a NFAT regulator protein encoded by ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) or DYRK6 (SEQ ID NO:9), or a homolog thereof.

The NFAT regulator protein of the invention can be produced by a variety of means known in the art, e.g. automated peptide synthesis or culturing a host cell comprising a recombinant vector, the recombinant vector comprising a nucleic acid sequence, the nucleic acid sequence comprising/encoding the NFAT regulator or a fragment or derivative thereof, wherein the host cell is cultured under conditions suitable for expression of the NFAT regulator.

The present invention further provides a system comprising an isolated cell comprising at least one heterologous NFAT regulator protein or fragment or derivative thereof, and/or at least one heterologous nucleic acid encoding a NFAT regulator protein or fragment or derivative thereof; and a monitoring agent used to monitor, detect, or measure electrical current across the plasma membrane of the cell. In one embodiment, the monitoring agent is an apparatus. In one embodiment, the electrical current is due to flux of cations or divalent ions across the cell. In one embodiment, the monitoring agent is used to monitor the effect of a test agent on intracellular calcium within the cell. In one embodiment, the monitoring agent is used to monitor, detect, or measure a calcium-entry mediated event.

The present invention further provides a system comprising a recombinant cell overexpressing at least one mammalian NFAT regulator protein or fragment or derivative thereof; and a monitoring agent used to monitor, detect, or measure a calcium-entry mediated event. In one embodiment, the NFAT regulator is encoded by ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO: 4), DYRK1B (SEQ ID NO: 5), DYRK2 (SEQ ID NO: 6), DYRK3 (SEQ ID NO: 7), DYRK4 (SEQ ID NO: 8) or DYRK6 (SEQ ID NO: 9).

The present invention further provides a recombinant cell comprising at least one heterologous NFAT regulator protein or fragment or derivative thereof, and/or at least one heterologous nucleic acid encoding a NFAT regulator protein or fragment or derivative thereof. In one embodiment, the recombinant cell overexpresses at least one mammalian NFAT regulator protein or fragment or derivative thereof.

The present invention further provides a recombinant cell overexpressing at least on mammalian NFAT regulator protein or fragment or derivative thereof.

The present invention further provides a method for identifying an agent for treating or

preventing a disease or disorder associated with calcium signaling. The method comprises assessing the effects of a test agent on an organism exhibiting the disease or disorder, and identifying the test agent as an agent for treating or preventing the disease or disorder if it modulates an activity, interaction, expression, or binding of at least one NFAT regulator protein or fragment thereof. In one embodiment, the disease or disorder is rheumatoid arthritis, inflammatory bowel disease, allogeneic or xenogeneic transplantation rejection, graft-versus-host disease, aplastic anemia, psoriasis, lupus erytematosus, inflammatory disease, MS, type I diabetes, asthma, pulmonary fibrosis, scleroderma, dermatomyositis, Sjogren's syndrome, postpericardiotomy syndrome, Kawasaki disease, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pemphigus vulgaris, autoimmune hemolytic anemia, idiopathic thrombopenia, chronic glomerulonephritis, Goodpasture's syndrome, Wegner's granulomatosis, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, uveitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's disease, ulcerative colitis, colitis/inflammatory bowel syndrome, Guilllain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, eczema, and autoimmune thyroiditis. Transplant graft rejections, acquired immunodeficiencies, common variable immunodeficiency, myocardial hypertrophy, severe combined immunodeficiency, dilated cardiomyopathy, excessive or pathological bone resorption, excessive adipocyte differentiation, obesity, or reactivation of latent viruses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show gene-dosage effect in store-operated Ca²⁺ entry (SOCE).

FIG. 1A shows a pedigree of patients with a defect in SOCE and CRAC channel function. Two male SCID patients (subject ID numbers 8 and 11; filled black squares) were born to consanguineous parents (subject ID numbers 35 and 36). For functional and genetic analysis, DNA and blood samples were obtained from all individuals shown in yellow or black. Half black squares or circles indicate heterozygous disease carriers as determined by phenotypic analysis. Double horizontal bars indicate consanguineous marriages, black boxes SCID disease, diagonal bars death of individuals. FIG. 1B shows reduced SOCE in T cells of both parents of CRAC deficient SCID patients that defines them as heterozygous carriers of the disease trait. T cells were stimulated with thapsigargin (TG) in the absence of extracellular Ca²⁺. The peak (upper panel) and rate (bottom panel) of Ca²⁺ influx were measured after readdition of 0.5 mM extracellular Ca²⁺. FIG. 1C shows reduced SOCE that phenotypically identifies 12/21 family members of the SCID patients as heterozygous disease trait carriers. Ca²⁺ influx was measured as described in B but using 0.2 mM extracellular Ca²⁺. Shown are the averages of Ca²⁺ influx rates from 4-5 experiments. Individual ID numbers correspond to those shown in FIG. 1A. Stars indicate heterozygous carriers as defined by influx rates below 2 nM/s (dotted red line). Co, healthy control; P, patient.

FIGS. 2A-2B show that a genome-wide RNAi screen identifies Drosophila Orai as a protein regulating NFAT translocation and store-operated Ca²⁺ entry. FIG. 2A shows that RNAi of dSTIM or dOrai inhibits dephosphorylation of NFAT. S2R+ cells stably transfected with NFAT1(1-460)-GFP were incubated for 4 days with double-stranded (ds) RNAi against dSTIM, dOrai or an irrelevant DNA sequence (mock). Cells were left unstimulated (−TG) or stimulated with thapsigargin (+TG) for 10 min, then lysed after stimulation with TG, and cell extracts were separated by SDS-PAGE and immunoblotted with antibodies against NFAT1. Dephosphorylation of NFAT is evidenced by more rapid migration (lower band) on SDS-PAGE. FIG. 2B shows that RNAi of either dSTIM or dOrai inhibits Ca²⁺ influx in S2R+ cells. Cells were left unstimulated (−TG) or stimulated with thapsigargin (+TG) for 10 min, then loaded with Fluo-4 and Fura-Red and analyzed for Ca²⁺ influx by flow cytometry. 1 μM thapsigargin was added at the indicated time. The top line in each panel shows RNAi for Gfp and the bottom line RNAi for dSTIM or dOrai. Decreased Ca²⁺ influx is indicated by the much reduced change in emission ratio following addition of thapsigargin.

FIGS. 3A-3C show that Orai1 is a transmembrane protein. FIG. 3A shows that Orai1 is highly conserved in eukaryotes. Shown is the sequence conservation in the first of four putative transmembrane regions (M1, underlined) of Orai1, which contains the R>W mutation (bold) found in the SCID patients. FIG. 3B shows membrane topology of Orai1. Hydropathy plots were calculated from the full-length amino acid sequence of human Orai1 (301 a.a., NP_(—)116179) using the Kyte-Doolittle algorithm with a window size of 19 amino acids. Three transmembrane domains (M2-M4) are predicted with a score>1.8; M1 has a score of ˜1.3. FIG. 3C shows schematic representation of the predicted membrane topology of Orai1, based on the hydropathy plot and immunocytochemistry data. The site of the R>W mutation in the SCID patients is indicated by a dark box. FIGS. 4A-4H show that expression of Orai1 restores CRAC channel function in SCID T cells. FIG. 4A shows activation of an inward current in an Orai^(WT)-complemented SCID T cell by passive store depletion with a pipette solution containing 8 mM BAPTA. At the indicated times, the 20 mM Ca²⁺ _(o) solution was replaced with a divalent free (DVF) solution. Enhanced current in the absence of divalent cations is a characteristic of CRAC channels and certain other Ca²⁺-selective channels. FIG. 4B shows the current-voltage (I-V) relation of currents in 20 mM Ca²⁺ _(o) (left) and in DVF solution (right) measured during voltage ramps from −100 to +100 mV. Data were collected at the times indicated by the arrows in 4A. Note that the Ca²⁺ current I-V relation is inwardly rectifying with a reversal potential >+90 mV. In DVF solution, the current reversed at ˜+50 mV. FIG. 4C shows that SCID T cells expressing mutant Orai1^(R>W), inward Ca²⁺ and Na⁺ currents fail to develop during passive store depletion by 8 mM BAPTA. FIG. 4D shows noise characteristics of the depotentiating Na⁺ current. Top graph shows the mean current at a constant holding potential of −100 mV. The dotted line indicates the zero current level (measured in 20 mM Ca²⁺+2 μM La³⁺). Variance was calculated from 100-ms segments of the Na⁺ current and plotted against mean current in lower panel. The data are fit by a straight line with a slope of 26 fA, giving a lower limit to the unitary current. FIG. 4E shows fast inactivation of the Ca²⁺ current in a SCID T cell expressing Orai^(WT). Fast inactivation was measured during 300-ms steps to −100 mV from a holding potential of +30 mV with 20 mM Ca²⁺ _(o). FIG. 4F shows blockade of the Ca²⁺ current by 2 μM La³⁺. After passive induction of the inward current in a SCID T cell expressing Orai^(WT), 2 μM La³⁺ was applied. The dotted line indicates the zero current level, determined from traces collected at the beginning of the experiment immediately following whole-cell break-in. FIG. 4G shows potentiation and blockade of I_(CRAC) by application, respectively, of low (5 μM) and high (40 μM) doses of 2-APB. FIG. 4H shows the summary of peak current densities in the indicated cell categories. Peak currents were measured during steps to −100 mV. Reconstitution with wild-type Orai1 thus reconstitutes a current with the expected characteristics of native CRAC channels. Cells transduced with Orai1^(WT) or Orai1^(R>W) were visually selected based on GFP-fluorescence; untransduced cells were GFP-negative.

FIGS. 5A-5D show that expression of Orai1 in fibroblasts from SCID patients restores store-operated Ca²⁺ influx. FIG. 5A shows inhibition of Ca²⁺ influx in Orai1^(WT) expressing SCID fibroblasts by 75 μM 2-APB. FIG. 5B shows potentiation of Ca²⁺ influx in Orai1^(WT)-expressing SCID fibroblasts by 3 μM 2-APB. FIGS. 5C-5D shows inhibition of Ca²⁺ influx in Orai1-expressing SCID fibroblasts by 2 μM La³⁺ added before (FIG. 5C) or after (FIG. 5D) readdition of 20 mM Ca²⁺. For each experiment, ˜15-20 GFP-positive fibroblasts were analyzed. Experiments were repeated at least three times for each protocol.

FIGS. 6A-6C show the NFAT regulatory domain and results of the genome-wide RNAi screen in Drosophila. FIG. 6A shows a schematic diagram of the N-terminal regulatory domain of NFAT1, showing the conserved phosphorylated serine motifs which are dephosphorylated upon stimulation (circles). Peptides corresponding to the SRR1, SP2 and SP3 motifs used for in vitro kinase assays are represented. Serine residues shown underlined have been identified to be phosphorylated in NFAT1 in vivo, and these are the residues mutated to alanine in the mutant SP2 and SP3 motifs. FIG. 6B shows that heterologously expressed NFAT is correctly regulated by Ca²⁺ and calcineurin in Drosophila S2R+ cells. Drosophila S2R+ cells were transfected with NFAT1-GFP expression vector. 48 hrs later, the cells were left untreated (Untr) or treated with thapsigargin (TG, 1 μM) for 30 min and lysates from the cells were analysed by immunoblotting (IB) with anti-NFAT1. P and deP refer to the migration positions of phosphorylated and dephosphorylated NFAT-GFP, respectively. FIG. 6C shows the tabulation of the results of the primary screen.

FIGS. 7A-7C shows screening of candidate kinases identified in the Drosophila S2R+ cell RNAi screen, for NFAT phosphorylation and identification of DYRK as a negative regulator of NFAT. FIG. 7A shows the ability of overexpressed mammalian homologs of the candidate kinases to directly phosphorylate the NFAT regulatory domain. FLAG-tagged mammalian homologues of selected Drosophila kinases were expressed in HEK293 cells, and immunopurified kinases were tested using an in vitro kinase assay for phosphorylation of GST-NFAT1 (1-415). Phosphorylation levels were assessed by autoradiography with either short (top panel) or long (middle panel) exposures. Expression of each kinase was verified by immunoblotting (IB) using an anti-FLAG antibody. Kinases tested are as follows: lane I, CK1α; lane 2, CK1ε; lane 3, Bub1; lane 4, STK38; lane 5, STK38L; lane 6, CDC42BPA; lane 7, ARAF; lane 8. PRKG1; lane 9, SGK; lanes 10 and 11, CSNKA1 and CSNKA2 (CKII isoforms); lane 12, SRPK1; lane 13, DYRK2; lane 14, ALS2CR7; lane 15, IRAK4. Bub1 was later dropped from our candidate list because of >10 predicted off-targets (Example 3).

FIG. 7B shows overexpression of DYRK2 blocks calcineurin-mediated dephosphorylation of NFAT1. Each kinase was co-transfected with NFAT-GFP into HEK293 cells; after 18 hrs cells were stimulated with 1 μM ionomycin in the presence of 2 mM CaCl₂. Lysates were immunoblotted using NFAT1 antibody. Relative expression levels of the kinases were determined by immunoblot using anti-FLAG antibody, and were identical to those represented in FIG. 6A (bottom panel). FIG. 7C shows depletion of endogenous DYRK1A potentiates NFAT activation. HeLa cells stably expressing Ha-tagged NFAT1-GFP were transfected with control siRNA or DYRK1A-specific siRNA. After 4 days cells were stimulated with 1 μM thapsigargin (TG) or 1 μM thapsigargin (TG) followed by 20 nM CsA for indicated times; lysates were immunoblotted for NFAT-GFP using anti-HA antibody (left). DYRK1A mRNA levels (right) were assessed after 3 and 4 days by real-time PCR. siControl, scrambled control siRNA; siDYRK1A, DYRK1A-specific siRNA. Results show the average and standard deviation of three independent experiments.

FIGS. 8A-8C show that DYRK2 inhibits NFAT-dependent reporter activity and endogenous IL-2 expression. FIG. 8A shows that overexpression of DYRK2 inhibits IL2 promoter-driven luciferase activity in stimulated Jurkat T cells. (The IL2 promoter is an example of a cytokine promoter whose activation exhibits a strong requirement for NFAT.) Exponentially growing Jurkat T cells were co-transfected with pRLTK (renilla luciferase, internal control), IL-2-pGL3 (IL-2-promoter driven firefly luciferase, experimental promoter) and empty vector or increasing amounts of wild type (WT) or kinase dead (KD) DYRK2 expression plasmids (5, 10, 15 and 20 μg). After 24 h cells untreated or stimulated with PMA and ionomycin for 6 h were analyzed for IL-2-promoter-driven luciferase activity. Firefly luciferase was normalized to renilla luciferase and fold induction calculated relative to IL-2 promoter activity measured in untreated cells. Results show the average and standard deviation of three independent experiments. FIG. 8B shows that overexpression of DYRK2 inhibits endogenous IL-2 expression in stimulated Jurkat T cells. Exponentially-growing Jurkat T cells were co-transfected with GFP and empty vector or increasing amounts of wild type (WT) or kinase dead (KD) DYRK2 expression plasmids (10, 20 and 30 μg). After 24 h cells untreated or stimulated with PMA and ionomycin for 6 h were evaluated for IL-2 expression in GFP+ cells by intracellular cytokine staining and flow cytometry. FIG. 8C shows quantification of the results shown in 8B. Results show the average and standard deviation of three independent experiments.

FIGS. 9A-9C shows that STIM proteins affect NFAT localization by altering store-operated Ca²⁺ influx. FIG. 9A shows the percent of cells with nuclear NFAT was quantified in three independent experiments after mock treatment or treatment with dsRNAs against dSTIM. Mean and standard deviations are plotted. 50-100 cells were analyzed for each experiment. FIG. 9B shows the effect of RNAi-mediated depletion of Drosophila STIM (dSTIM) on NFAT phosphorylation status. Lysates made from unstimulated or thapsigargin (TG)-stimulated S2R+ cells were examined by immunoblotting with antibody against NFAT1. The cells were mock-treated or treated for 4 days with dsRNAs targeting dSTIM. FIG. 9C shows intracellular Ca²⁺ levels, analyzed by flow cytometry, in S2R+ cells depleted with dSTIM or novel gene candidates from the confirmatory screen. GFP dsRNA was used as a control for non-specific effects caused by dsRNA treatment. After 30 sec of basal [Ca²+]_(I) measurement, 1 μM thapsigargin was added (arrow) and [Ca²+]_(I) measurements were continued for a further 5 min. Depletion of dSTIM almost completely abolishes thapsigargin-triggered, that is store-operated, Ca²⁺ influx.

FIGS. 10A-10B shows the phylogenetic relation between different members of the DYRK family in Drosophila and in humans, and the expression pattern of human DYRKs in Jurkat T cells. FIG. 10A shows the phylogenetic tree of DYRK family kinases using distance-based methods (neighbour joining). The left-hand side figures show the homology relationships between Drosophila CG40478 and human DYRK 2, 3; Drosophila CG4551 (smi35A) and human DYRK 4; Drosophila CG7826 (mnb) and human DYRK1 A, B (top); as computed by the program Tcoffee. In the right-hand side figures, the orthologue bootstrap value for CG40478-DYRK2 is higher than for CG40478-DYRK3 (top). Therefore, DYRK2 is an orthologue of CG40478 (the genes diverged by a speciation event), while DYRK3 may be a paralogue (the genes diverged by a duplication event). The calculations of the ortholog bootstrap values were performed with Orthostrapper. FIG. 10B shows expression of DYRK family members in Jurkat T cells. Expression level of mammalian DYRK mRNAsin Jurkat T cells was estimated by RT-PCR analysis. Primers correspond to:

(SEQ ID NO: 10) DYRK1A sense: AGTTCTGGGTATTCCACCTGCTCA (SEQ ID NO: 11) DYRK1A anti-sense: TGAAGTTTACGGGTTCCTGGTGGT; (SEQ ID NO: 12) DYRK2 sense: TCCACCTTCTAGCTCAGCTTCCAA, (SEQ ID NO: 13) DYRK2 anti-sense: TGGCAACACTGTCCTCTGCTGAAT; (SEQ ID NO: 14) DYRK1B sense: GCCAGCTCCATCTCCAGTTCT, (SEQ ID NO: 15) DYRK1B anti-sense: CACAATATCGGTTGCTGTAGCGGT; (SEQ ID NO: 16) DYRK3 sense: TGCAATCCTTCTGAACCACCTCCA, (SEQ ID NO: 17) DYRK3 anti-sense: GCTGTTCTACCTTCATCTCACCTCCA; (SEQ ID NO: 18) DYRK4 sense: AGGCTGTCATCACTCGAGCAGAAA, (SEQ ID NO: 19) DYRK4 anti-sense: AGTCCTGCTGATCACCTGAATGCT; (SEQ ID NO: 20) DYRK6 sense: GCCGATGAGCATATGGCAAACACA, (SEQ ID NO: 21) DYRK6 anti-sense: TACCCACTGCAGAAGGCTGGTTTA.

FIGS. 11A-11I show the nucleotide sequences for NFAT regulator genes. FIG. 11A shows the nucleotide sequence ORAI1 (NM_(—)032790; SEQ ID NO: 1). FIG. 11B shows the nucleotide sequence for ORAI2 (BC069270; SEQ ID NO:2). FIG. 11C shows the nucleotide sequence for ORAI3 (NM_(—)152288; SEQ ID NO:3). FIG. 11D shows the nucleotide sequence for DYRK1A (NM_(—)001396; SEQ ID NO:4). FIG. 11E shows the nucleotide sequence for DYRK1B (NM_(—)004714; SEQ ID NO:5). FIG. 11F shows the nucleotide sequence for DYRK2 (NM_(—)003583; SEQ ID NO:6). FIG. 11G shows the nucleotide sequence for DYRK3 (NM_(—)003582; SEQ ID NO:7). FIG. 11H shows the nucleotide sequence for DYRK4 (NM_(—)003845; SEQ ID NO:8). FIG. 11I shows the nucleotide sequence for DYRK6 (NM_(—)005734; SEQ ID NO:9).

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention relate to the characterization of genes regulating NFAT activity, for example, via Store-Operated Calcium Entry (SOCE) or via modulation of NFAT phosphorylation. In particular, to the discovery of an essential component of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Accordingly, aspects of the invention relate to novel regulators of NFAT activity, particularly with regard to modulation of NFAT activity in T cells. Aspects of the invention also relate to methods to screen for novel agents that modulate NFAT activity. Aspects of the invention further relate to methods to screen for agents that modulate the activity of the NFAT regulators of the present invention. The invention further provides methods to screen for agents that modulate the NFAT regulators of the present invention by means of modulating intracellular calcium.

NFAT Genes and Proteins

By NFAT protein (nuclear factor of activated T cells) is meant a member of a family of transcription factors comprising the members NFAT1, NFAT2, NFAT3 and NFAT4, with several isoforms. Any other NFAT protein whose activation is calcineurin dependent is also meant to be included. NFAT proteins can be, e.g., mammalian proteins, e.g., human or murine. NFAT1, NFAT2 and NFAT4 are expressed in immune cells, e.g., T lymphocytes, and play a role in eliciting immune responses. NFAT proteins are involved in the transcriptional regulation of cytokine genes, e.g., IL-2, IL-3, IL-4, TNF-alpha and IFN-gamma, during the immune response.

The conserved regulatory domain of NFAT is an N-terminal region of NFAT which is about 300 amino acids in length. The conserved regulatory domain of murine NFAT1 is a region extending from amino acid residue 100 through amino acid residue 397, of human NFAT1 is a region extending from amino acid residue 100 through 395, of human NFAT2 is a region extending from amino acid residue 106 through 413, of human NFAT2b is a region extending from amino acid residue 93 through 400, of human NFAT3 is a region extending from amino acid residue 102 through 404, and of human NFAT4 is a region extending from amino acid residue 97 through 418. The conserved regulatory domain is moderately conserved among the members of the NFAT family, NFAT1, NFAT2, NFAT3 and NFAT4. The conserved regulatory region binds directly to calcineurin. The conserved regulatory region is located immediately N-terminal to the DNA-binding domain (amino acid residues 398 through 680 in murine NFAT1, amino acid residues 396 through 678 in human NFAT1, amino acid residues 414 through 696 in human NFAT2, amino acid residues 401 through 683 in human NFAT2b, amino acid residues 405 through 686 in human NFAT3, and amino acid residues 419 through 700 in human NFAT4).

Store Operated Calcium Entry

SOCE is one of the main mechanisms to increase intracellular cytoplasmic free Ca²⁺ concentrations ([Ca²⁺]i) in electrically non-excitable cells. Ca²⁺ elevations are a crucial signal transduction mechanism in virtually every cell. The tight control of intracellular Ca²⁺, and its utility as a second messenger, is emphasized by the fact that [Ca²+]i levels are typically 70-100 nM while extracellular Ca²⁺ levels ([Ca²+]ex) are 10⁴-fold higher, ˜1-2 mM. The immediate source of Ca²⁺ for cell signaling can be either intracellular or extracellular (FIG. 1). Intracellular Ca²⁺ is released from ER stores by inositol 1,4,5-triphosphate (IP3), or other signals, while extracellular Ca²⁺ enters the cell through voltage-gated, ligand-gated, store-operated or second messenger-gated Ca²⁺ channels in the plasma membrane. In electrically non-excitable cells such as lymphocytes, the major mechanism for Ca²⁺ entry is store-operated Ca²⁺ entry, a process controlled by the filling state of intracellular Ca²⁺ stores. Depletion of intracellular Ca²⁺ stores triggers activation of membrane Ca²⁺ channels with specific electrophysiological characteristics, which are referred to as calcium release-activated Ca²⁺ (CRAC) channels (Parekh and Putney, Jr. 2005, Physiol Rev 85:757). Ca²⁺ release activated Ca²⁺ (CRAC) channels. The electrophysiological characteristics of CRAC channels have been studied intensively, but the molecular nature of the channel itself and the mechanisms of its activation remain unknown. One definition of CRAC channels holds that depletion of intracellular Ca²⁺ stores is both necessary and sufficient for channel activation without direct need for increases in [Ca²+]i, inositol phosphates IP3 or IP4, cGMP or cAMP (Parekh and Penner. 1997, Physiol Rev. 77:901). Biophysically, CRAC current is defined, amongst other criteria, by its activation as a result of ER Ca²⁺ store depletion, its high selectivity for Ca²⁺ over monovalent (Cs⁺, Na⁺) cations, a very low single channel conductance, a characteristic I-V relationship with pronounced inward rectification and its susceptibility to pharmacological blockade for instance by La³+ and 2-APB (100 μM), respectively (Parekh and Putney, Jr. 2005, Physiol Rev 85:757; Lewis, 2001, Annu Rev Immunol 19:497).

Candidate genes for SOCE and CRAC. The molecular nature of the CRAC channel remains completely unknown. The most widely investigated candidate genes for the CRAC channel have been the >25 mammalian homologues of the Drosophila photoreceptor TRP (Transient Receptor Potential) gene. But most TRP proteins form non-specific cation channels and even those that show some preference for divalent cations do not exhibit all of the key biophysical hallmarks of the CRAC channel when beterologously expressed (Clapham, 2003. Nature 426:517). Until recently, TRPV6 was the most promising CRAC channel candidate gene because some of its biophysical features overlapped with that of CRAC. But while TRPV6, like CRAC, selectively conducts Ca²⁺, it is not activated by store depletion, a defining characteristic of the CRAC channel. Knockdown studies using RNAi to suppress TRPV6 expression and our studies using T cells from TRPV6−/− mice showed no defect in SOCE or ICRAC in the absence of TRPV6 (Kahr, et al. 2004. J Physiol 557:121; Kepplinger, et al. Neither CaT1 nor TRPC3 proteins contribute to CRAC of T lymphocytes. Manuscript in preparation). Thus, neither TRPV6 nor any other gene has been confirmed to be involved in SOCE or CRAC channel activity.

Mechanisms of SOCE and CRAC Channel Activation.

The mechanism by which CRAC channels are activated is equally unclear. Depletion of intracellular Ca²⁺ stores is necessary for CRAC activation but how the information about reduced Ca²⁺ concentrations in the ER is conveyed to the CRAC pore is not known. Three main models have been proposed but no consensus has been reached (Parekh and Putney, Jr. 2005, Physiol Rev 85:757). (i) The “conformational coupling model” postulates a conformational change of a molecule at the surface of the ER which then binds to the CRAC channel; (ii) The “secretion coupling model” suggests that (constitutively active) CRAC channels reside in intracytoplasmic vesicles that fuse to the plasma membrane upon store depletion; (iii) The “Calcium influx factor (CIF) model” predicts a soluble small molecule, which activates Ca²⁺ influx through CRAC channels when CIF is released into the cytoplasm of stimulated cells.

Stromal interaction molecule 1 (STIM1). Recent evidence suggests that STIM1 plays an important role in store operated Ca²⁺ entry and CRAC channel function. Three independent RNAi screens by Roos et al. (2005, J Cell Biol 169:435), Liou et al. (2005, Curr Biol 15:1235) and by our group (see Example 2 below) have found that suppression of STIM expression by RNAi impairs Ca²⁺ influx in Drosophila melanogaster S2 cells as well as mammalian cells (FIG. 5). STIM1 is a type I transmembrane protein which was initially characterized as a stromal protein promoting the expansion of pre-B cells and as a putative tumor suppressor (Oritani, et al. 1996. J Cell Biol 134:771; Sabbioni, et al. 1997. Cancer Res 57:4493). The human gene for STIM1 is located on chromosome 11p15.5 which is believed to contain genes associated with a number of pediatric malignancies, including Wilms tumor (Parker et al. 1996, Genomics 37:253). STIM1 contains a Ca²⁺ binding EF hand motif and a sterile α-motif (SAM) domain in its ER/extracellular region, a single membrane-spanning domain, and two predicted cytoplasmic coiled-coil regions (Manji et al. 2000, Biochim Biophys Acta 1481:147). Domain structure and genomic organization are conserved in a related gene called STIM2, which differs from STIM1 mainly in its C-terminus (Williams et al. 2002, Biochim Biophys Acta 1596:131). STIM1 is able to homodimerize or heterodimerize with STIM2 (Williams et al. 2002 supra). Expressed in the ER, its C-terminal region is located in the cytoplasm whereas the N-terminus resides in the lumen of the ER, as judged by glycosylation and phosphorylation studies (Maji et al. 2000 supra; Williams et al. 2002 supra). A minor fraction of STIM1 is located in the plasma membrane. Although RNAi mediated suppression of STIM1 expression interferes with SOCE and CRAC channel function, STIM1 is unlikely to be a Ca²⁺ channel itself. Rather it is thought that STIM1 may sense Ca²⁺ levels in the ER via its EF hand (Putney, Jr. 2005. J Cell Biol 169:381; Marchant, 2005, Curr Biol 15:R493). Consistent with the conformational coupling model of store-operated Ca²⁺ influx, STIM1 could act as a key adapter protein, which physically bridges the space between ER and plasma membrane, and thus directly connects sensing of depleted Ca²⁺ stores to store-operated Ca²⁺ channels in the plasma membrane (Putney, Jr. 2005. supra; Putney, Jr. 1986, Cell Calcium 7:1).

NFAT Regulators

As used herein, the term “NFAT regulators” is used to refer to the proteins (NFAT regulator proteins), and the encoding genes (NFAT regulator genes) which regulate NFAT activity. The methods of the present invention are intended to include use of homologues, analogues, isoforms (e.g. alternative splice variants), derivatives, and functional fragments of the NFAT regulators described herein. Preferably, homologues of NFAT regulator proteins have at least 70%, more preferably, 80%, and more preferably 90% amino acid identity to those specifically identified herein.

NFAT Regulator Proteins

In one preferred embodiment, the NFAT regulator proteins of the present invention are encoded by the ORAI genes. Previous to the discoveries upon which the present invention is based, the function of the ORAI genes was unknown. ORAI1 nucleic acid sequence corresponds to GenBank accession number NM_(—)032790, ORAI2 nucleic acid sequence corresponds to GenBank accession number BC069270 and ORAI3 nucleic acid sequence corresponds to GenBank accession number NM_(—)152288. As used herein, ORAI refers to any one of the ORAI genes, e.g., ORAI1, ORAI2, ORAI3.

In one embodiment, the NFAT regulator proteins of the present invention are encoded by the DYRK genes. Previous to the discoveries upon which the present invention is based, the DYRK genes were not known to regulate NFAT activity or function. DYRK1A is encoded by several nucleic acid isoforms including GenBank accession numbers NM_(—)001396, NM_(—)101395, NM_(—)130436, NM_(—)130437, and NM_(—)130438. DYRK1Bis encoded by multiple nucleic acid isoforms including GenBank accession numbers NM_(—)004714, NM_(—)006483, and NM_(—)006484. DYRK2 is encoded by GenBank accession numbers including NM_(—)003583 and NM_(—)006482. DYRK3 is encoded by GenBank accession numbers including NM_(—)001004023 and NM_(—)003582. DYRK4 is encoded by GenBank accession number NM_(—)003845. DYRK6, also known as HIPK3, is encoded by GenBank accession number NM_(—)005734.

In one embodiment, the NFAT regulator proteins of the present invention are encoded by the genes listed in Table I.

The term “fragment” or “derivative” when referring to a NFAT regulator protein means proteins or polypeptides which retain essentially the same biological function or activity in at least one assay as the native NFAT regulator proteins. For example, the NFAT regulator fragments or derivatives of the present invention maintain at least about 50% of the activity of the native proteins, preferably at least 75%, more preferably at least about 95% of the activity of the native proteins, as determined e.g. by a calcium influx assay described in Example 1.

Fragments or derivatives as the term is used herein can include competitors of the native NFAT regulators with respect to a particular NFAT regulator domain activity. However, the fragment or derivative shows an overall similarity to NFAT regulators in other areas as explained herein.

The term fragment, as used herein, refers to a fragment of the NFAT regulator protein, or nucleic acid sequence, wherein the (encoded) protein retains at least one biological activity of the full length NFAT regulator protein. The term fragment and functional fragment are used herein interchangeably. A fragment of a sequence contains less nucleotides or amino acids than the corresponding full length sequences, wherein the sequences present are in the same consecutive order as is present in the full length sequence. As such, a fragment does not contain internal insertions or deletions of anything (e.g. nucleic acids or amino acids) in to the portion of the full length sequence represented by the fragment. This is in contrast to a derivative, which may contain internal insertions or deletions within the nucleic acids or amino acids that correspond to the full length sequence, or may have similarity to full length coding sequences.

A derivative may comprise the same or different number of nucleic acids or amino acids as full length sequences. The term derivative, as used herein with respect to an NFAT regulator protein, includes NFAT regulator proteins, or fragments thereof, which contain one or more modified amino acids. e.g. chemically modified, or modification to the amino acid sequence (substitution, deletion, or insertion). Such modifications should substantially preserve at least one biological activity of the NFAT regulator protein. Such biological activity is readily determined by a number of assays known in the art, for example, a calcium influx assay described below in Example 1. By way or nonlimiting example, a derivative may be prepared by standard modifications of the side groups of one or more amino acid residues of the NFAT regulator protein, its analog, or a functional fragment thereof, or by conjugation of the NFAT regulator protein, its analogs or fragments, to another molecule e.g. an antibody, enzyme, receptor, etc., as are well known in the art. Accordingly, “derivatives” as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention. Derivatives may have chemical moieties such as carbohydrate or phosphate residues. Such a derivativization process should preserve at least one biological activity of the NFAT regulator protein. Derivatives can be made for convenience in expression, for convenience in a specific assay, to enhance detection, or for other experimental purposes. Derivatives include dominant negatives, dominant positives and fusion proteins.

Antibodies

In one embodiment, the invention provides antibodies to the NFAT regulators of the present invention. Antibodies can be prepared that will bind to one or more particular domains of a peptide of the invention and can be used to modulate NFAT regulator gene or protein activity.

Moreover, administration of an antibody against an NFAT regulator protein or fragment or derivative thereof, preferably monoclonal or monospecific, to mammalian cells (including human cells) can reduce or abrogate NFAT induced transcription of immune system associated genes, thus serving to treat hyperactivity or inappropriate activity of the immune system. Administration of an activating antibody against an NFAT regulator protein or fragment or derivative thereof, e.g. an Orai protein, may serve to treat hypoactivity of the immune system by activating NFAT and thereby inducing transcription of immune response associated genes. Administration of an antibody against an NFAT regulator protein or fragment or derivative thereof, e.g., a DYRK protein, may serve to treat hypoactivity of the immune system by activating NFAT and thereby inducing transcription of immune response associated genes.

The present invention also relates to antibodies that bind a protein or peptide encoded by all or a portion of the NFAT regulator nucleic acid molecule, as well as antibodies which bind the protein or peptide encoded by all or a portion of a variant nucleic acid molecule. For instance, polyclonal and monoclonal antibodies which bind to the described polypeptide or protein, or fragments or derivatives thereof, are within the scope of the invention.

Antibodies of this invention can be produced using known methods. An animal, such as a mouse, goat, chicken or rabbit, can be immunized with an immunogenic form of the protein or peptide (an antigenic fragment of the protein or peptide which is capable of eliciting an antibody response). Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art. The protein or peptide can be administered in the presence of an adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassays can be used with immunogen as antigen to assess the levels of antibody. Following immunization, anti-peptide antisera can be obtained, and if desired, polyclonal antibodies can be isolated from the serum. Monoclonal antibodies can also be produced by standard techniques which are well known in the art (Kohler and Milstein, Nature 256:4595-497 (1975); Kozbar et al., Immunology Today 4:72 (1983); and Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Such antibodies are useful as diagnostics for the intact or disrupted gene, and also as research tools for identifying either the intact or disrupted gene.

As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody to NFAT regulator proteins may be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) to thereby isolate immunoglobulin library members that bind to NFAT regulator proteins. Kits for generating and screening phage display libraries are commercially available from, e.g., Dyax Corp. (Cambridge, Mass.) and Maxim Biotech (South San Francisco, Calif.). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display libraries can be found in the literature.

Polyclonal sera and antibodies may be produced by immunizing a suitable subject, such as a rabbit, with NFAT regulator proteins (preferably mammalian; more preferably human) or an antigenic fragment thereof. The antibody titer in the immunized subject may be monitored over time by standard techniques, such as with ELISA, using immobilized marker protein. If desired, the antibody molecules directed against NFAT regulator proteins may be isolated from the subject or culture media and further purified by well-known techniques, such as protein A chromatography, to obtain an IgG fraction.

Fragments of antibodies to NFAT regulator proteins may be produced by cleavage of the antibodies in accordance with methods well known in the art. For example, immunologically active F(ab′) and F(ab′)₂ fragments may be generated by treating the antibodies with an enzyme such as pepsin. Additionally, chimeric, humanized, and single-chain antibodies to NFAT regulator proteins, comprising both human and nonhuman portions, may be produced using standard recombinant DNA techniques. Humanized antibodies to NFAT regulator proteins may also be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes.

NFAT Associated Diseases

The methods of the present invention can also be utilized to treat, or identify agents useful in treatment of, conditions and diseases associated with NFAT disregulation/disfunction and/or Calcium signaling. Such diseases include, without limitation, immune system diseases involving hyperactivity or inappropriate activity of the immune system, e.g., acute immune diseases, chronic immune diseases and autoimmune diseases Examples of such diseases include rheumatoid arthritis, inflammatory bowel disease, allogeneic or xenogeneic transplantation rejection (organ, bone marrow, stem cells, other cells and tissues), graft-versus-host disease, aplastic anemia, psoriasis, lupus erytematosus, inflammatory disease, MS, type I diabetes, asthma, pulmonary fibrosis, scleroderma, dermatomyositis, Sjogren's syndrome, postpericardiotomy syndrome, Kawasaki disease, Hashimoto's thyroiditis, Gravest disease, myasthenia gravis, pemphigus vulgaris, autoimmune hemolytic anemia, idiopathic thrombopenia, chronic glomerulonephritis, Goodpasture's syndrome, Wegner's granulomatosis, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, uveitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's disease, ulcerative colitis, colitis/inflammatory bowel syndrome, Guilllain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, eczema, and autoimmune thyroiditis. Transplant graft rejections can result from tissue or organ transplants. Graft-versus-host disease can result from bone marrow or stem cell transplantation. Immune system diseases involving hypoactivity of the immune system include, e.g., immunodeficiency diseases including acquired immunodeficiencies, such as HIV disease, and common variable immunodeficiency (CVID).

The methods of the present invention can also be utilized to treat or identify agents useful in treatment of conditions and diseases that are not immune mediated, but which nevertheless involve the Ca²⁺-calcineurin-mediated activation of NFAT, e.g. a protein-protein interaction between calcineurin and NFAT. Examples include myocardial hypertrophy, dilated cardiomyopathy, excessive or pathological bone resorption, excessive adipocyte differentiation, obesity, and reactivation of latent human herpesvirus-8 or other viruses. Further, the methods of the present invention can be utilized to treat, or identify agents useful in the treatment of, conditions that involve a dysfunction of cellular Ca²⁺ signaling, attributable to altered function of an NFAT regulator protein, wherein, the dysfunction of Ca²⁺ signaling causes a disease or disorder at least in part through its effects on other Ca²⁺ dependent pathways in addition to the Ca²⁺-calcineurin-NFAT pathway, or wherein the dysfunction of Ca²⁺ signaling acts largely through such other pathways and the changes in NFAT function are ancillary.

Severe Combined Immunodeficiency

One NFAT associated disease/disorder is Severe Combined Immunodeficiency (SCID). SCID is a group of congenital immune disorders caused by failed or impaired development and/or function of both T and B lymphocytes. A rare disease with an estimated prevalence of 1 per 100,000 population, SCID can be caused by mutations in more than 20 different genes. Mutations in the common γ chain (cγ) of the interleukin 2 (IL-2), IL-4, -7, -9 and -15 receptors leading to X-1 inked SCID account for 50% of all cases. Approximately 10% of all SCID cases are due to a variety of rare mutations in genes important for T and B cell development or function, especially signal transduction (CD3ε and γ, ZAP-70, pS61ck, CD45, JAK3, IL-7Rα chain). Due to the low incidence of these mutations and small family sizes, classical positional cloning is usually not possible for most of these SCID diseases and mutations were often found in known signal transducing genes by functional analysis of T cells followed by sequencing of candidate genes. Scientifically, SCID disease has been of extraordinary value for the elucidation of T cell and B cell function, highlighting the consequences of gene dysfunction in the immune system.

In one embodiment, the invention relates to a method to diagnose unexplained immunodeficiency in a subject comprising comparison of a nucleotide sequence corresponding to a gene from the subject comprising the NFAT regulators of the present invention to wild type sequence of that gene, wherein alteration of the nucleotide sequence of the gene from the wild type sequence indicates that the alteration in the gene is responsible for the immunodeficiency. In one embodiment, the alteration in the gene is a mutation in a splice site. In one embodiment, the alteration in the gene is a nonsynonymous mutation. In one embodiment, the unexplained immunodeficiency is associated with defects in regulation of NFAT activity.

In one embodiment, the comparison is accomplished by way of obtaining a biological sample from the subject, sequencing the DNA in the biological sample, and electronically aligning the DNA sequence obtained from the biological sample to a wild type sequence.

In one embodiment, a comparison is accomplished by way of obtaining a DNA sample, processing the DNA sample such that the DNA is available for hybridization, combining the DNA with nucleotide sequences complementary to the nucleotide sequence of a NFAT regulator of the present invention under conditions appropriate for hybridization of the probes with complementary nucleotide sequences in the DNA sample, thereby producing a combination; and detecting hybridization in the combination, wherein absence of hybridization in the combination is indicative of alteration in the nucleotide sequence in the gene.

Method to Screen for Agents that Modulate NFA T Regulator Function

In one embodiment, the present invention relates to methods to screen for agents that alter NFAT regulator expression or function. In one embodiment, the present invention relates to methods to screen for agents that alter the function of the NFAT regulator proteins of the present invention. NFAT regulator function may be altered as to the modulation of CRAC channel activation. NFAT regulator function may be altered as to the modulation of NFAT phosphorylation. NFAT regulator function may be altered as to modulation of NFAT subcellular localization. NFAT regulator function may be altered as to modulation of free intracellular calcium levels. NFAT regulator function may be altered as to modulation of calcineurin activity. In one embodiment, alter or modulate refers to upregulation or enhancement of activity. In one embodiment, alter or modulate refers to downregulation or inhibition.

As used herein, the term “NFAT regulator genes” is used to refer to the genes identified by the methods of the present invention that regulate NFAT activity, including by way of SOCE, by way of direct phosphorylation of NFAT or by other means as described in example 2. The NFAT regulator genes of the present invention include: ORAI1, ORAI2, ORAI3, the DYRK genes including DYRK1A, DYRK1B, DYRK2, DYRK3 DYRK4 and DYRK6 and the genes disclosed in Table I in Example 3. In one preferred embodiment, the NFAT regulator genes of the present invention are ORAIs, e.g., ORAI1, ORAI2, and ORAI3. The NFAT regulator genes and/or their encoded protein products, modulate the activity of NFAT either directly or indirectly.

As used herein, the term “modulates” refers the effect an agent, including a gene product, has on another agent, including a second gene product. In one embodiment, an agent that modulates another agent upregulates or increases the activity of the second agent. In one embodiment, an agent that modulates another agent downregulates or decreases the activity of the second agent.

One example of an NFAT regulator detected through the RNAi screening described herein is calcineurin. The role of calcineurin in NFAT signaling was previously known. Specifically, calcineurin dephosphorylates and activates NFAT, and therefore is a positive regulator.

Calcineurin serves to illustrate the relationship between altered expression of a regulator and altered NFAT signaling: Overexpression of calcineurin leads to increased activation of NFAT in standard assays; conversely, diminished expression of calcineurin, as in the RNAi screen detailed below in Example 1, leads to a decrease in NFAT activation. Calcineurin also illustrates that altered activity of a regulator, by an agent, is reflected in altered NFAT signaling. Thus, cyclosporin A and FK506 are calcineurin inhibitors when complexed with their cytoplasmic binding proteins (cyclophilin A and FKBP12, respectively), and the inhibitory action of these compounds on calcineurin can be detected, for example, by examining the effect of cyclosporin A or FK506 on NFAT localization in cells stimulated with thapsigargin, or in T cells stimulated physiologically through the T cell receptor.

An assay for an agent that affects an NFAT regulator need not directly involve NFAT. Thus, a number of agents that alter the activity of calcineurin, for example, the PVIVIT peptide and its derivatives, the CsA-cyclophilin A complex, and the FK506-FKBP12 complex, can be assayed by examining their binding to calcineurin; and the calcineurin autoinhibitory peptide can be assayed by examining its effect on dephosphorylation of substrates other than NFAT.

Positive regulators of NFAT are known to act at other stages of the Ca²⁺-calcineurin-NFAT signaling pathway. For example, Orai1 and STIM1 contribute to the elevation of cytoplasmic [Ca²⁺], and thereby elicit activation of calcineurin and subsequently of NFAT. Here again, agents that decrease expression of Orai1 or STIM1 (e.g., RNAi reagents, as shown herein for both Orai1 and STIM1; and as shown for dStim and STIM1 in Roos et al (2005) J Cell Biol 169, 435-445; Liou et al (2005) Current Biology 15, 1235-1241) can be recognized either by their effects on NFAT activation (e.g., NFAT dephosphorylation or intracellular localization) or on other parameters diagnostic of the function of the NFAT regulators in question (e.g., cytoplasmic Ca²⁺ levels). Agents that inhibit function of the Ca²⁺-calcineurin-NFAT signaling pathway by affecting one or more NFAT regulator proteins, for example agents that inhibit Ca²⁺ influx through the CRAC channel (e.g., La³⁺, Gd³⁺, 2-APB) are likewise readily detected. The inhibitory agents that are known at present, however, are not entirely selective, which is the reason that the assays described herein constitute a valuable tool for the discovery of agents that target the NFAT modulator proteins of this pathway more selectively.

The present invention is also inclusive of negative regulators of Ca²⁺-calcineurin-NFAT signaling. These include, for example, DYRK-family kinases, casein kinase-1 isoforms, and glycogen synthase kinase (GSK-3). Inhibition of the expression of these negative regulators, for example by RNAi treatment, or inhibition of their activity, for example by treatment with an agent that inhibits enzyme activity (e.g., the casein kinase inhibitor CKI-7; Li⁺ as a GSK-3 inhibitor), in each case can be detected using an assay that monitors an aspect of NFAT activation.

The invention relates to screening methods (also referred to herein as “assays”) for identifying modulators, i.e., candidate compounds or agents (e.g., proteins, peptides, peptidomimetics, peptoids, oligonucleotides (such as siRNA or anti-sense RNA), small non-nucleic acid organic molecules, small inorganic molecules, or other drugs) that bind to NFAT regulator proteins, or to NFAT, have an inhibitory (or stimulatory) effect on, for example, NFAT regulator gene expression or protein activity, NFAT gene expression or protein activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of an NFAT regulator-interacting protein (e.g. a NFAT regulator substrate) or a NFAT-interacting protein (e.g. a NFAT substrate). Such interacting proteins can include Ca²⁺ and other subunits of calcium channels, proteins that interact with one or more Orai proteins, e.g., additional CRAC channel subunits or CRAC channel modulatory proteins. Compounds thus identified can be used to modulate the activity of target gene products (e.g., NFAT regulator polypeptides, NFAT polypeptides) either directly or indirectly in a therapeutic protocol, to elaborate the biological function of the target gene product, or to identify compounds that disrupt the normal interactions of the target gene or gene product. Identification of a blocking agent or inhibitor of an NFAT regulator gene or an encoded product can be carried out using the screening methods of this invention and other methods known in the art. Compounds that affect NFAT regulator expression or activity can be identified as described herein or using other methods known in the art. The modulator compounds can be novel, compounds not previously identified as having any type of activity as a calcium channel modulator, or a compound previously known to modulate calcium channels, but that is used at a concentration not previously known to be effective for modulating calcium influx. The modulator can also be a modulator compound for NFAT regulators other than CRAC channel components.

The term “agent” or “compound” as used herein and throughout the specification means any organic or inorganic molecule, including modified and unmodified nucleic acids such as antisense nucleic acids, RNAi, such as siRNA or shRNA, peptides, peptidomimetics, receptors, ligands, and antibodies.

Compounds that inhibit the activity or expression of an NFAT regulator are useful in the treatment of disorders involving cells that express an NFAT regulator. Particularly relevant disorders are those involving hyperactivity or inappropriate activity of the immune system or hypoactivity of the immune system, as further described herein.

Cells or tissues affected by these disorders can be used in screening methods, e.g., to test whether an agent that modulates expression or activity of an NFAT regulator can reduce proliferation of affected cells, alleviate abnormal SOCE function, or alleviate abnormal NFAT activity. Other cells useful in the screening methods of the present invention are cells that exhibit store-operated calcium entry, which include insect cells, e.g., Drosophila cells (e.g., Schneider 2 or S2 cells), human embryonic kidney (HEK) cells, neuronal or nervous system cells, e.g., SHSY5Y neuroblastoma cells and PC12 cells, rat basophilic leukemia (RBL) cells, and immune system cells, e.g., primary T cells from mammals such as human or mouse, lymphocytes such as T lymphocytes, including Jurkat cells. Cells derived from the knock out or transgenic animals described below may be useful. Cells derived from immunodeficient patients, e.g., patients described in Example 1, including T cells and fibroblasts, may be useful in the methods of the present invention.

As used herein, the term “recombinant cell” is used to refer to a cell with exogenous and/or heterologous nucleic acid incorporated within, either incorporated stably so as to remain incorporated in clonal expansion of the cells, or introduced transiently into a cell (or a population of cells). The nucleic acid may contain, for example, an NFAT regulator gene or it's mRNA, or its complementary (antisense) strand, or an shRNA or siRNA, or any fragment or derivative of the foregoing. The nucleic acid may comprise genomic DNA of NFAT regulator proteins, fragments, or derivative thereof. The nucleic acid can comprise corresponding coding and non-coding mRNA or its complementary (anticoding) strand, which can be employed to regulate expression of the corresponding mRNA, e.g. corresponding short nucleotides of shRNA or siRNA. The nucleic acid can result in altered expression (e.g. over expression or underexpression) of at least one NFAT regulator protein or its mRNA or antisense. It may also result in the expression of a NFAT regulator protein functional fragment or derivative otherwise not expressed in the recipient cell.

Test Compounds

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but that nevertheless remain bioactive; see, e.g., Zuckermann, et al., 1994 J. Med. Chem. 37: 2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA. 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; and Ladner supra.).

The compounds that can be screened by the methods described herein include, but are not limited to, any small molecule compound libraries derived from natural and/or synthetic sources, small non-nucleic acid organic molecules, small inorganic molecules, peptides, peptoids, peptidomimetics, oligonucleotides (e.g., siRNA, antisense RNA, aptamers such as those identified using SELEX), and oligonucleotides containing synthetic components.

The test compounds can be administered, for example, by diluting the compounds into the medium wherein the cell is maintained, mixing the test compounds with the food or liquid of a test animal (see below), topically administering the compound in a pharmaceutically acceptable carrier on the test animal, using three-dimensional substrates soaked with the test compound such as slow release beads and the like and embedding such substrates into the test animal, intracranially administering the compound, parenterally administering the compound.

A variety of other reagents may also be included in the mixture. These include reagents such as salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding and/or reduce non-specific or background interactions, etc. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The language “pharmaceutically acceptable carrier” is intended to include substances capable of being coadministered with the compound and which allow the active ingredient to perform its intended function of preventing, ameliorating, arresting, or eliminating a disease(s) of the nervous system. Examples of such carriers include solvents, dispersion media, adjuvants, delay agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media and agent compatible with the compound may be used within this invention.

The compounds can be formulated according to the selected route of administration. The addition of gelatin, flavoring agents, or coating material can be used for oral applications. For solutions or emulsions in general, carriers may include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride, potassium chloride among others. In addition intravenous vehicles can include fluid and nutrient replenishers, electrolyte replenishers among others.

Preservatives and other additives can also be present. For example, antimicrobial, antioxidant, chelating agents, and inert gases can be added (see, generally, Remington's Pharmaceutical Sciences, 16th Edition, Mack, 1980).

Test Assays for Agents that Modulate NFAT Activity

Another aspect of the invention relates to a method to screen for regulators of free intracellular Ca²⁺ levels, calcineurin activation and NFAT localization in cells as described in Examples 1 through 3. In one embodiment, a recombinant vector encoding a fusion protein comprising the entire NFAT regulatory domain or a functional fragment or derivative thereof, and an operably linked reporter protein (for determining subcellular localization of the regulatory domain, e.g. GFP or an antigenic epitope) is transfected into cells, i.e. test cells. Test cells transfected with the vector are contacted with the test agent. After a period of time, e.g., 48-72 hours, the test cells are scored for subcellular localization of the NFAT-reporter fusion protein. Scoring may be accomplished by way of automated microscopy, as in the examples, or by way of manual microscopy, e.g., fluorescent microscopy, confocal microscopy. Secondary test assays include calcium influx detection assays. If the test agent has an effect on intracellular localization of the expression product of the recombinant vector, this is indicative that it modulates NFAT regulator function.

In one embodiment, the cells also express an exogenous (e.g. heterologous or homologous) NFAT regulator protein, or fragment or derivative thereof, and/or exhibit altered expression of a NFAT regulatory protein or fragment or derivative thereof, achieved with the tools/methods described herein.

In one embodiment, the test cells are resting cells wherein NFAT is normally localized to the cytoplasm. Nuclear localization, or partial nuclear localization in excess of that observed in untreated control cells, of the NFAT-reporter fusion protein in the resting test cell indicates that the test agent successfully activated NFAT activity.

In one embodiment, the test cells are stimulated cells, wherein intracellular Ca²⁺ stores are depleted and store-operated Ca²⁺ entry is activated and NFAT is localized to the nucleus. Ca²⁺ store depletion may be accomplished, for example, by means of contacting the test cells with thapsigargin or ionomycin. The test cells may be stimulated prior to, concurrently with or subsequent to contacting the test cells with the test agent. Cytoplasmic localization, or a reduction in nuclear localization compared to that observed in control cells, of the NFAT-reporter fusion protein in the stimulated test cell indicates that the test agent successfully inhibited NFAT activation.

A reporter gene which encodes a reporter protein to be operably linked to nucleotide sequences encoding the NFAT regulatory domain, any reporter gene for general use is satisfactory provided that its localization in the cell can be assessed either directly or indirectly in the context of the fusion protein. For example, the reporter can be any protein whose localization can be detected by staining with a labeled antibody, or a protein epitope such as a haemagglutinin or myc epitope, or green fluorescent protein (GFP) or one of its variants. In one preferred embodiment, the reporter protein is GFP. The NFAT protein in the fusion protein may be full length or may comprise the regulatory domain, particularly the calcineurin and CK1 docking sites and the conserved serine rich regions (SRR) and serine-proline (SP) repeat motifs.

Another aspect of the invention relates to methods for identifying an agent for treating or preventing a disease or disorder associated with calcium signaling. In one embodiment, the method comprises assessing the effects of a test agent on an organism that exhibits the disease or disorder, or exhibits at least one phenotype associated with the disease or disorder. The test agent is identified as an agent for treating or preventing the disease or disorder if it modulates an activity, interaction, expression or binding of at least one NFAT regulator protein, fragment, or derivative thereof. In one embodiment, the NFAT regulator protein, fragment, or derivative thereof is expressed either endogenously or exogenously in cells of the organism. Appropriate methods of administration of the test agent and assessment of effects can be determined by the skilled practitioner.

Test Assays for Agents that Modulate Calcium Levels

In monitoring the effect of a test agent on intracellular calcium in any of the screening/identification methods provided herein, a direct or indirect evaluation or measurement of cellular (including cytosolic and intracellular organelle or compartment) calcium and/or movement of ions into, within or out of a cell, organelle, or portions thereof (e.g., a membrane) can be conducted. A variety of methods are described herein and/or known in the art for evaluating calcium levels and ion movements or flux. The particular method used and the conditions employed can depend on whether a particular aspect of intracellular calcium is being monitored. For example, as described herein, reagents and conditions are known, and can be used, for specifically evaluating store-operated calcium entry, resting cytosolic calcium levels and calcium levels and uptake by or release from intracellular organelles. The effect of test agent on intracellular calcium can be monitored using, for example, a cell, an intracellular organelle or storage compartment, a membrane (including, e.g., a detached membrane patch or a lipid bilayer) or a cell-free assay system. Generally, monitoring the effect of a test agent on intracellular calcium involves contacting a test agent with or exposing a test agent to (1) a protein (and/or nucleic acid, or portion(s) thereof, encoding a protein) involved in modulating intracellular calcium (in particular, a protein provided herein) and/or (2) a cell, or portion(s) thereof (e.g., a membrane or intracellular structure or organelle) that may or may not contain a protein (and/or nucleic acid, or portion(s) thereof, encoding a protein) involved in modulating intracellular calcium. A cell can be one that exhibits one or more aspects of intracellular Ca²⁺ modulation, such as, for example, store-operated calcium entry. Before, during and/or after the contacting of test agent, a direct or indirect assessment of intracellular calcium can be made. An indirect assessment can be, for example, evaluation or measurement of current through an ion transport protein (e.g., a store-operated calcium channel or a Ca²⁺-regulated ion channel), or transcription of a reporter protein operably linked to a calcium-sensitive promoter. A direct assessment can be, for example, evaluation or measurement of intracellular (including cytosolic and intracellular organelle) calcium.

The assessment of intracellular calcium is made in such a way as to be able to determine an effect of an agent on intracellular calcium. Typically, this involves comparison of intracellular calcium in the presence of a test agent with a control for intracellular calcium. For example, one control is a comparison of intracellular calcium in the presence and absence of the test agent or in the presence of varying amounts of a test agent. Thus, one method for monitoring an effect on intracellular calcium involves comparing intracellular calcium before and after contacting a test agent with a test cell containing a protein that modulates intracellular calcium, or comparing intracellular calcium in a test cell that has been contacted with test agent and in a test cell that has not been contacted with test agent (i.e., a control cell). Generally, the control cell is substantially identical to, if not the same as, the control cell, except it is the cell in the absence of test agent. A difference in intracellular calcium of a test cell in the presence and absence of test agent indicates that the agent is one that modulates intracellular calcium.

Another method for monitoring an effect on intracellular calcium involves comparing intracellular calcium of a test cell and a control cell that is substantially similar to the test cell (e.g., comparing a cell containing a protein (and/or nucleic acid encoding a protein) involved in intracellular calcium signaling, such as the proteins provided herein), and a cell that does not contain, or that contains lower levels of, the particular protein involved in modulating intracellular calcium signaling. Thus, for example, if the test cell containing the protein involved in intracellular calcium modulation is a recombinant cell generated by transfer of nucleic acid encoding the protein into a host cell, then one possible control cell is a host cell that has not been transfected with nucleic acid encoding the protein or that has been transfected with vector alone. Such a cell would be substantially similar to the test cell but would differ from the test cell essentially only by the absence of the introduced nucleic acid encoding the protein. Thus, a control cell may contain, e.g., endogenously, the particular protein involved in modulating intracellular calcium, in which case the test cell would contain higher levels of (or overexpress) the particular protein.

It may also be useful to experimentally reduce the endogenous expression or functional levels of a particular protein (e.g. by inhibition of protein expression or function) to identify an agent that modulates intracellular calcium by targeting that particular protein. Expression of an NFAT regulator protein can be reduced in a cell by known experimental methods such as by targeting expression at the nucleic acid level, e.g. siRNA or shRNA treatment, to thereby reduce expression of functional protein. Systems which comprise such a cell which have reduced, or completely inhibited, expression of NFAT regulator are included in this invention. Such systems may further contain an exogenous (e.g. homologous or heterologous) nucleic acid molecule encoding one or more mammalian NFAT regulator proteins, or a portion thereof, in expressible form.

The type of control comparison described above, where endogenous expression/functional levels of a particular protein are reduced in a cell, is particularly useful when trying to identify an agent that specifically modulates intracellular calcium via an effect on, or modulation of, a particular protein (and/or nucleic acid, or portion(s) thereof, encoding a particular protein). Thus, for example, if there is no detectable or substantial difference in intracellular calcium in the test (non-modified) versus control (reduced endogenous expression/function) cells in the presence of the agent, the agent likely does not mediate its effect on intracellular calcium via the particular protein (or nucleic acid encoding the protein). A detectable or substantial difference in intracellular calcium in the test versus control cells in the presence of the test agent, indicates the test agent may be a candidate agent that specifically modulates intracellular calcium via an effect on or modulation of the particular protein. A candidate agent can be subjected to further control assays to compare intracellular calcium in test cells in the presence and absence of test agent or to compare intracellular calcium in control cells in the presence and absence of test agent, which can aid in determination of whether a candidate agent is an agent that modulates intracellular calcium. An assessment of intracellular calcium conducted to monitor the effect of test compound on intracellular calcium can be made under a variety of conditions. Conditions can be selected to evaluate the effect of test compound on a specific aspect of intracellular calcium. For example, as described herein, reagents and conditions are known, and can be used, for specifically evaluating store-operated calcium entry, resting cytosolic calcium levels and calcium levels of and calcium uptake by or release from intracellular organelles. For example, as described herein, calcium levels and/or calcium release from the endoplasmic reticulum can directly be assessed using mag-fura 2, endoplasmic reticulum-targeted aequorin or cameleons. One method for indirect assessment of calcium levels or release is monitoring intracellular cytoplasmic calcium levels (for example using fluorescence-based methods) after exposing a cell to an agent that effects calcium release (actively, e.g., IP₃, or passively, e.g., thapsigargin) from the organelle in the absence of extracellular calcium. Assessment of the effect of the test agent/compound on concentrations of cations or divalent cations within the cell, or of ion influx into the cell, can also be used to identify a test agent as an agent that modulates intracellular calcium.

Resting cytosolic calcium levels, intracellular organelle calcium levels and cation movement may be assessed using any of the methods described herein or known in the art (see, e.g., descriptions herein of calcium-sensitive indicator-based measurements, such as fluo-3, mag-fura 2 and ER-targeted aequorin, labeled calcium (such as ⁴⁵Ca²⁺)-based measurements, and electrophysiological measurements). Particular aspects of ion flux that may be assessed include, but are not limited to, a reduction (including elimination) or increase in the amount of ion flux, altered biophysical properties of the ion current, and altered sensitivities of the flux to activators or inhibitors of calcium flux processes, such as, for example, store-operated calcium entry. Reagents and conditions for use in specifically evaluating receptor-mediated calcium movement and second messenger-operated calcium movement are also available.

In particular embodiments of the methods for screening for or identifying agents that modulate intracellular calcium, the methods are conducted under conditions that permit store-operated calcium entry to occur. Such conditions are described herein and are known in the art. Test agents can be contacted with a protein and/or nucleic acid encoding a protein (such as the proteins and nucleic acids provided herein) involved in modulating intracellular calcium and/or a cell (or portion thereof containing such a protein (or nucleic acid) under these appropriate conditions. For example, in conducting one method for screening for an agent that modulates intracellular calcium under conditions selected for evaluating store-operated calcium entry, intracellular calcium levels of test cells are monitored over time using a fluorescent calcium indicator (e.g., FLUO-4). Store-operated calcium entry into the cells is detected depending on the specific indicator used as, e.g. an increase in fluorescence, a decrease in fluorescence, or a change in the ratio of fluorescence intensities elicited by excitation using light of two different wavelengths. in response to conditions under which store-operated calcium entry occurs. The methods for eliciting the fluorescence signal for a specific calcium indicator and for interpreting its relation to a change in free calcium concentration are well known in the art. The conditions include addition of a store-depletion agent, e.g., thapsigargin (which inhibits the ER calcium pump and allows discharge of calcium stores through leakage) to the media of cell that has been incubated in Ca²⁺-free buffer, incubation with thapsigargin for about 5-15 minutes, addition of test compound (or vehicle control) to the media and incubation of the cell with test agent for about 5-15 minutes, followed by addition of external calcium to the media to a final concentration of about 1.8 mM. By adding thapsigargin to the cell in the absence of external calcium, it is possible to delineate the transient increase in intracellular calcium levels due to calcium release from calcium stores and the more sustained increase in intracellular calcium levels due to calcium influx into the cell from the external medium (i.e., store-operated calcium entry through the plasma membrane that is detected when calcium is added to the medium). Because the fluorescence-based assay allows for essentially continuous monitoring of intracellular calcium levels during the entire period from prior to addition of thapsigargin until well after addition of calcium to the medium, not only can “peak” or maximal calcium levels resulting from store-operated calcium entry be assessed in the presence and absence of test agent, a number of other parameters of the calcium entry process may also be evaluated, as described herein. For example, the kinetics of store-operated calcium entry can be assessed by evaluation of the time required to reach peak intracellular calcium levels, the up slope and rate constant associated with the increase in calcium levels, and the decay slope and rate constant associated with the decrease in calcium levels as store-operated calcium entry discontinues. Any of these parameters can be evaluated and compared in the presence and absence of test agent to determine whether the agent has an effect on store-operated calcium entry, and thus on intracellular calcium. In other embodiments, store-operated calcium entry can be evaluated by, for example, assessing a current across a membrane or into a cell that is characteristic of a store-operated calcium entry current (e.g., responsiveness to reduction in calcium levels of intracellular stores) or assessing transcription of a reporter construct that includes a calcium-sensitive promoter element. In particular embodiments, a test agent is identified as one that produces a statistically significant difference. E.g., at least a 30% difference in any aspect or parameter of store-operated calcium entry relative to control (e.g., absence of compound, i.e., vehicle only).

Generally, a test agent is identified as an agent, or candidate agent, that modulates intracellular calcium if there is a detectable effect of the agent on intracellular calcium levels and/or ion movement or flux, such as a detectable difference in levels or flux in the presence of the test agent. In particular embodiments, the effect or differences can be substantial or statistically significant.

Test Assays for Agents that Modulate NFAT Regulator Activity

In one embodiment, an assay is a cell-based assay in which a cell that expresses an NFAT regulator protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate NFAT regulator activity is determined. Determining the ability of the test compound to modulate NFAT regulator activity can be accomplished by monitoring, for example, changes in calcium flux in the cell or by testing downstream effects of modulating calcium flux such activation or IL-2 expression. Methods of testing such downstream effects are known in the art and include modulation of cell proliferation and cell growth. For example, a compound that decreases the number of NFAT regulator molecules in a cell or affects the function of an NFAT regulator channel may decrease cellular proliferation. Alternatively, transcription of genes requiring NFAT transactivation may be monitored.

U.S. Pat. Application No. 20040002117 discloses known gene targets of NFAT and teaches methods to identify further genes transcribed due to activity of NFAT. Detection of transcription or protein expression of NFAT target genes may be useful in the methods of the present invention. Ablation of induced expression of NFAT target genes in the presence of a test agent indicates that the test agent is effective in inhibiting NFAT regulator activity, where the NFAT regulator is a positive regulator of NFAT. Conversely, expression of NFAT target genes above basal levels in the presence of a test agent, in otherwise unstimulated conditions, indicates that the test agents is effective in inhibiting a negative regulator of NFAT.

In some cases, the cell used in such assays does not normally express the NFAT regulator of interest (e.g. a channel protein). By way of non-limiting example, a cell such as a Xenopus oocyte or immune system cell or derivative thereof can be engineered to expresses a recombinant NFAT regulator protein, biologically active portion or derivative thereof. In general, recombinant expression that results in increased expression of the NFAT regulator compared to a corresponding cell that does not express recombinant NFAT regulator, is referred to as “overexpression” of the NFAT regulator. Alternatively, the cell can be of mammalian origin. The cell can also be a cell that expresses the NFAT regulator of interest (e.g. a calcium channel) but in which such NFAT regulator activity can be distinguished from other NFAT regulator (e.g. calcium channel) activity, for example, by comparison with controls. The ability of the test compound to modulate NFAT regulator binding to a compound, e.g., an NFAT regulator substrate, or to bind to NFAT regulator can also be evaluated. This can be accomplished, for example, by coupling the compound, e.g., the substrate, with a radioisotope or enzymatic label such that binding of the compound, e.g., the substrate, to NFAT regulator can be determined by detecting the labeled compound, e.g., substrate, in a complex. Alternatively, NFAT regulator could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate NFAT regulator binding to an NFAT regulator substrate in a complex. For example, compounds (e.g., NFAT regulator substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

An example of a screening assay for a compound that specifically modulates activity of an NFAT regulator polypeptide is as follows. Incubate a cell that expresses the NFAT regulator polypeptide of interest (e.g., a Jurkat cell or an HEK293 cell) with a test compound for a time sufficient for the compound to have an effect on transcription or activity (e.g., for at least 1 minute, 10 minutes, 1 hour, 3 hours, 5 hours, or 24 or more hours. Such times can be determined experimentally. The concentration of the test compound can also be varied (e.g., from 1 nM-100 μM, 10 nM to 10 μM or, 1 nM to 10 μM). Inhibition of calcium influx in the presence and absence of the test compound is then assayed using methods known in the art. For example, fura-2, Indo-1, Fluo-3, or Rho-2 can be used to assay calcium flux. Other methods can be used as assays of inhibition. For example, a test compound is considered to have, or suspected of, having a significant impact on influx if any one or more of the following criteria are met:

-   -   a. there is direct inhibition of increased [Ca²+]i as measured         by a calcium indicator.     -   b. there is a direct inhibition of I_(CRAC) as measured by patch         clamp;     -   c. there is inhibition of downstream signaling functions such as         calcineurin activity, NFAT subcellular localization, NFAT         phosphorylation, and/or cytokine, e.g., IL-2, production; or     -   d. there are modifications in activation-induced cell         proliferation, differentiation and/or apoptotic signaling         pathways.

Direct testing of the effect of a test compound on an activity of a specific NFAT regulator polypeptide can be accomplished using, e.g., patch clamping to measure I_(CRAC). This method can be used in screening assays as a second step after testing for general effects on calcium influx or as a second step after identifying a test compound as affecting expression of an NFAT regulator mRNA or polypeptide. Alternatively, direct testing can be used as a first step in a multiple step assay or in single step assays.

The ability of a compound (e.g., an NFAT regulator substrate) to interact with the NFAT regulator with or without the labeling of any of the interactants can be evaluated. For example, a microphysiometer can be used to detect the interaction of a compound with NFAT regulator without the labeling of either the compound or the NFAT regulator (McConnell et al., 1992, Science 257:1906-1912). As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and NFAT regulator polypeptide.

In yet another embodiment, a cell-free assay is provided in which a NFAT regulator protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the NFAT regulator protein or biologically active portion thereof is evaluated. Preferred biologically active portions of the NFAT regulator proteins to be used in assays of the present invention include fragments or derivatives that participate in interactions with other signaling molecules, or fragments or derivatives that interact directly with NFAT.

Cell-free assays involve preparing a reaction mixture of the target gene protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., using fluorescence resonance energy transfer (FRET) (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No. 4,868,103). A fluorophore label is selected such that a first ‘donor’ label's emission spectrum overlaps with the absorption spectrum of a second, ‘acceptor’ molecule, which then fluoresces on excitation of the donor, if the labels are in close proximity, due to transfer of energy. Alternately, the ‘donor’ protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the ‘acceptor’ molecule label may be differentiated from that of the ‘donor’. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ‘acceptor’ molecule label in the assay is increased over the emission when binding does not occur, or when, e.g., binding is prevented by the excess of unlabelled competitor protein. A FRET binding event can be conveniently measured, in comparison to controls, through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).

Assays which monitor assembly of the protein complex in cells or in cell free assays may also be used.

In another embodiment, determining the ability of the NFAT regulator protein to bind to a target molecule can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance” or “BIA” detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal that can be used as an indication of real-time reactions between biological molecules.

In one embodiment, the target gene product, e.g., NFAT regulator polypeptide or the test substance, is anchored onto a solid phase. The target gene product/test compound complexes anchored on the solid phase can be detected at the end of the reaction. In general, the target gene product can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein.

It may be desirable to immobilize an NFAT regulator, an anti-NFAT regulator antibody or its target molecule to facilitate separation of complexed from non-complexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to an NFAT regulator protein, or interaction of an NFAT regulator protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/NFAT regulator fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione Sepharose™ beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or NFAT regulator protein, and the mixture incubated under conditions conducive for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of NFAT regulator binding or activity determined using standard techniques.

Other techniques for immobilizing either NFAT regulator protein or a target molecule on matrices include using conjugation of biotin and streptavidin. Biotinylated NFAT regulator protein or target molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemicals).

To conduct the assay, the non-immobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).

This assay is performed utilizing antibodies reactive with NFAT regulator protein or target molecules but which do not interfere with binding of the NFAT regulator protein to its target molecule. Such antibodies can be derivatized to the wells of the plate, and unbound target or NFAT regulator protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with NFAT regulator protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the NFAT regulator protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. In such an assay, the reaction products are separated from unreacted components, by any of a number of standard techniques, including, but not limited to: filtration; differential centrifugation (see, for example, Rivas and Minton, 1993, Trends Biochem. Sci. 18:284-7); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and immunoprecipitation (see, for example, Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see, e.g., Heegaard, 1998, J. Mol. Recognit. 11: 141-8; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. Sci. Appl. 699:499-525). Further, fluorescence resonance energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.

The assay can include contacting the NFAT regulator protein or biologically active portion thereof with a known compound that binds NFAT regulator to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an NFAT regulator polypeptide, wherein determining the ability of the test compound to interact with an NFAT regulator protein includes determining the ability of the test compound to preferentially bind to NFAT regulator or biologically active portion thereof, or to modulate the activity of a target molecule, as compared to the known compound.

To the extent that NFAT regulator can, in vivo, interact with one or more cellular or extracellular macromolecules, such as proteins, inhibitors of such an interaction are useful. Such interacting molecules include Ca²⁺ and subunits of the calcium channel complex as well as signaling molecules that directly interact with the channel, such as kinases, phosphatases and adapter proteins, can be used to identify inhibitors. For example, a preformed complex of the target gene product and the interactive cellular or extracellular binding partner product is prepared such that either the target gene products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Pat. No. 4,109,496 that utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt target gene product-binding partner interaction can be identified. Alternatively, an NFAT regulator polypeptide can be used as a “bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., 1993, Cell 72:223-232; Madura et al., 1993, J. Biol. Chem. 268:12046-12054; Bartel et al., 1993, Biotechniques 14:920-924; Iwabuchi et al., 1993, Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, that bind to or interact with NFAT regulator (“NFAT regulator -binding proteins” or “NFAT regulator-bp”) and are involved in NFAT regulator activity. Such NFAT regulator-bps can be activators or inhibitors of signals by the NFAT regulator proteins or NFAT regulator targets as, for example, downstream elements of an NFAT regulator-mediated signaling pathway, e.g., NFAT target gene expression or activity.

Modulators of NFAT regulator expression can also be identified. For example, a cell or cell free mixture is contacted with a candidate compound and the expression of an NFAT regulator mRNA or protein evaluated relative to the level of expression of an NFAT regulator mRNA or protein in the absence of the candidate compound. Methods to dectect expression or evaluate expression level are well known to the skilled artisan. When expression of an NFAT regulator mRNA or protein is greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NFAT regulator mRNA or protein expression. Alternatively, when expression of NFAT regulator mRNA or protein is less (i.e., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NFAT regulator mRNA or protein expression. The level of NFAT regulator mRNA or protein expression can be determined by methods described herein for detecting an NFAT regulator mRNA or protein.

A modulating agent can be identified using a cell-based or a cell-free assay, and the ability of the agent to modulate the activity of a NFAT regulator protein can be confirmed in vivo, e.g., in an animal such as an animal model for a disease (e.g., an animal with leukemia or autoimmune disease or an animal harboring a xenograft from an animal (e.g., human) or cells from a cancer resulting from a leukemia or other lymphocytic disorder, or cells from a leukemia or other lymphocytic disorder cell line.

This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., a NFAT regulator -modulating agent, an antisense NFAT regulator nucleic acid molecule, a NFAT regulator -specific antibody, or a NFAT regulator -binding partner) in an appropriate animal model (such as those described above) to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.

Animal models that are useful include animal models of leukemia and autoimmune disorders. Examples of such animal models are known in the art and can be obtained from commercial sources, e.g., the Jackson Laboratory (Bar Harbor, Me.) or generated as described in the relevant literature. Examples of animals useful for such studies include mice, rats, dogs, cats, sheep, rabbits, and goats. Other useful animal models include, without limitation, those for other disorders of Ca²⁺-NFAT signaling or of Ca²⁺ signaling, e.g., for myocardial hypertrophy, dilated cardiomyopathy, excessive or pathological bone resorption, excessive adipocyte differentiation, obesity, and reactivation of latent human herpesvirus-8 or other viruses, as discussed elsewhere in this document.

Systems

Also provided herein are systems for use in identifying an agent that modulates one or more of the following: a NFAT protein, a NFAT regulator protein, and intracellular or cytoplasmic calcium. Such a system includes a cell, or portion(s) thereof, containing one or more proteins, e.g., NFAT regulator proteins of the present invention, or fragments or derivative thereof, e.g., ORAI proteins or fragments or derivatives thereof. In one embodiment, the proteins are exogenous (heterologous or homologous) to the cell. In one embodiment, the cell contains an exogenous (e.g. heterologous or homologous) nucleic acid encoding a NFAT regulator protein or fragment or derivative thereof. In one embodiment, the system further contains a monitoring agent used to monitor, detect or measure electrical current across the plasma membrane of the cell. Many such monitoring agents are known in the art. The term “monitoring agent” is also meant to include any apparatus used for such monitoring.

In particular embodiments of the systems, the protein(s) involved in modulating intracellular calcium are contained in cells. The cells can be isolated cells or cell cultures that endogenously express such protein(s) or recombinantly express such proteins as described above with respect to the methods for identifying agents, e.g. a recombinant cell overexpressing at least one NFAT regulator protein or fragment or derivative thereof. Systems in which the cells recombinantly express the proteins can be such that the cells are isolated cells or cell cultures or are contained within an animal, in particular, a non-human animal, e.g., a non-human mammal.

The proteins (and/or nucleic acids encoding proteins) or cells (or portions thereof) of the system can be contained in a medium that contains an agent that provides for passive or active intracellular calcium store reduction or depletion (e.g., thapsigargin and other agents described herein or known in the art) and/or that contains a molecule or molecules that facilitate monitoring or measurement of intracellular calcium and/or calcium movement. Such molecules include fluorescent (or otherwise labeled) calcium indicators, trivalent cations, divalent cations other than calcium and calcium-buffering agents, e.g., calcium chelators.

Recombinant Cells

Aspects of the invention further relate to recombinant cells used in the assays described in the methods discussed herein. In one aspect, the invention also encompasses any recombinant cells described herein. In one embodiment, the recombinant cell comprises at least one exogenous (heterologous or homologous) NFAT regulator protein or fragment or derivative thereof. The recombinant cell may also further comprise at least one exogenous (heterologous or homologous) nucleic acid encoding a NFAT regulator protein or fragment or derivative thereof. The NFAT regulator protein may be of mammalian origin. The recombinant cell may over express the NFAT regulator protein or fragment or derivative thereof. This overexpression may result from expression of an exogenous (heterologous or homologous) NFAT regulator protein (e.g. from an exogenous nucleic acid) or may result from over expression of native/endogenous NFAT regulator protein.

Transgenic Animals

The invention provides non-human transgenic animals that are engineered to overexpress an NFAT regulator, ectopically express an NFAT regulator, express reduced levels of an NFAT regulator, express a mutant NFAT regulator, or be knocked out for expression of an NFAT regulator. Such animals and cell lines derived from such animals are useful for studying the function and/or activity of an NFAT regulator protein and for identifying and/or evaluating modulators of NFAT regulator activity. An animal that overexpresses an NFAT regulator polypeptide is useful, e.g., for testing the effects of candidate compounds for modulating the activity of the NFAT regulator polypeptide and assessing the effect of the compound in vivo.

As used herein, a “transgenic animal” is a non-human animal, in general, a mammal, for example, a rodent such as a rat or mouse, in which one or more of the cells of the animal include a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA or a rearrangement, e.g., a deletion of endogenous chromosomal DNA, which is in most cases integrated into or occurs in the genome of the cells of a transgenic animal. A transgene can direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal; other transgenes, e.g., a knockout, reduce expression. Thus, a transgenic animal can be one in which an endogenous NFAT regulator gene has been altered by, e.g., by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a transgene of the invention to direct expression of an NFAT regulator protein to particular cells. A transgenic founder animal can be identified based upon the presence of an NFAT regulator transgene in its genome and/or expression of NFAT regulator mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding an NFAT regulator protein can further be bred to other transgenic animals carrying other transgenes.

NFAT regulator proteins or polypeptides can be expressed in transgenic animals or plants, e.g., a nucleic acid encoding the protein or polypeptide can be introduced into the genome of an animal. In preferred embodiments the nucleic acid is placed under the control of a tissue specific promoter, e.g., a milk or egg specific promoter, and recovered from the milk or eggs produced by the animal. Suitable animals are mice, pigs, cows, goats, and sheep.

In one non-limiting example, a mouse is engineered to express an NFAT regulator polypeptide using a T cell-specific promoter such as an LCK promoter using methods known in the art (e.g., Zhang et al., 2002, Nat. Immunol. 3:749-755). In an alternative example, a mouse is engineered with a tissue-specific knockdown of an NFAT regulator mRNA and protein, e.g., by Cre-lox mediated recombination, where expression of the recombinase is under control of a tissue-specific promoter. Engineered animals can be identified using known methods of identifying the presence of a transgene in cells and by assaying a cell sample (e.g., T cells) for the overexpression or underexpression of the NFAT regulator (for example, using immunocytochemistry) or by assaying calcium flux in a cell from the sample. Such transgenic animals are useful, e.g., for testing compounds for their ability to inhibit NFAT regulator-mediated cell proliferation.

The invention also includes a population of cells from a transgenic animal. Methods of developing primary, secondary, and immortal cell lines from such animals are known in the art.

Pharmaceutical Compositions

For therapeutic applications, peptides and nucleic acids of the invention, the antibodies to the NFAT regulators or the agents identified by the screening methods of the present invention, e.g., small molecules, siRNAs, shRNAs, may be suitably administered to a subject such as a mammal, particularly a human, alone or as part of a pharmaceutical composition, comprising the peptide, nucleic acid, antibody or agent together with one or more acceptable carriers thereof and optionally other therapeutic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The pharmaceutical compositions of the invention include those suitable for oral, rectal, nasal, topical, e.g, including buccal and sublingual, mucosal or parenteral, e.g., including subcutaneous, intramuscular, intravenous and intradermal administration. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well know in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa. (17th ed. 1985).

Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets optionally may be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Compositions suitable for topical administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Application of the subject therapeutics often will be local, so as to be administered at the site of interest. Various techniques can be used for providing the subject compositions at the site of interest, such as injection, use of catheters, trocars, projectiles, pluronic gel, stents, sustained drug release polymers or other device which provides for internal access. Where an organ or tissue is accessible because of removal from the patient, such organ or tissue may be bathed in a medium containing the subject compositions, the subject compositions may be painted onto the organ, or may be applied in any convenient way. Systemic administration of a nucleic acid using lipofection, liposomes with tissue targeting (e.g. antibody) may also be employed.

It will be appreciated that actual preferred amounts of a given peptide or nucleic acid of the invention, or of an antibody or agent identified by the screening methods of the present invention, used in a given therapy will vary to the particular active peptide or nucleic acid or agent being utilized, the particular compositions formulated, the mode of application, the particular site of administration, the patient's weight, general health, sex, etc., the particular indication being treated, etc. and other such factors that are recognized by those skilled in the art including the attendant physician or veterinarian. Optimal administration rates for a given protocol of administration can be readily determined by those skilled in the art using conventional dosage determination tests.

Various embodiments of the invention are further illustrated in the following examples. All references made to other publications or disclosures throught this document are incorporated by reference herein.

Example 1 Identification of Ca²⁺ Release Activated Ca²⁺ (CRAC) Channel Gene, ORAI1, in SCID Patients Materials and Methods: Case Reports

Detailed case reports of the two SCID patients investigated in this study have been described (Feske 1996, 2000).

Cell Lines and Reagents

T cell lines were established from peripheral blood lymphocytes of two patients and 21 family members and grown as described⁴⁸. Foreskin fibroblasts from the newborn SCID patient 2 and a healthy newborn (Hs27 cell line, ATCC, Manassas, Va.) were immortalized by retroviral transduction with a telomerase expression plasmid (hTERT, generous gift of S. Lessnick, DFCI, Boston, Mass.). The macrophage-hemocyte-like Drosophila cell line S2R+was grown in Schneider's medium with 10% fetal calf serum (Invitrogen) according to standard protocols. Thapsigargin was purchased from LC Biochemicals (Woburn, Mass.), Charybdotoxin (CTX) and 2-aminoethoxydiphenylborate (2-APB) from Sigma (St. Louis, Mo.).

Single Nucleotide Polymorphism (SNP) Array Based Linkage Analysis

Genomic DNA of SCID patients and 21 relatives was prepared from peripheral blood mononuclear cells using genomic DNA Maxi prep kits (Qiagen). Genotyping was performed at the SNP Genotyping Center (Broad Institute, Cambridge, Mass.) and the Harvard Partners Center for Genetics and Genomics (Boston, Mass.), using “GeneChip” Human Mapping 10K Arrays (Xba 142 2.0, Affymetrix, Santa Clara, Calif.) with an average SNP heterozygosity of 0.38 and a mean intermarker density of 258 kb. This platform allowed for simultaneous genotyping of more than 10,000 SNPs in the human genome. For parametric linkage analysis, data were converted into “Linkage” format using “Compare Linkage”⁴⁹. Mendelian genotype errors inconsistent with the parental genotypes were detected and set to missing genotypes. Multipoint parametric linkage analysis was performed to compute LOD scores at each SNP position using Allegro⁵⁰. To confirm linkage, we reanalyzed the SNP data using Genehunter 2.1r6⁵¹ and Merlin⁵² obtaining very similar results. For parametric analysis, a disease allele frequency of 0.001, a penetrance value of 0.99 and a phenocopy of 0.01 were used for all the pedigrees. Parametric linkage analyses were carried out using recessive and dominant models of inheritance, respectively. For the “recessive” model, haplotypes from both patients, their parents, unaffected brother and grandparents (individuals 8, II, 35, 36, 37, 38, 39, 63, 64 in FIG. 1A) were analyzed assuming an autosomal recessive mode of inheritance for the SCID disease with both SCID patients being homozygous for a common disease-causing mutation. The predicted maximum log₁₀ of the odds ratio (LOD) score from this analysis was ˜1.9 (i.e. −log₁₀[0.25×0.25×0.25×0.75]). For the “dominant” model, 12 family members with reduced store-operated Ca²⁺ entry were defined as “affected”, i.e. carriers of a dominantly acting mutation, and their SNP haplotypes compared to those of 8 healthy family members with normal store-operated Ca²⁺ entry. The predicted maximum LOD score from this analysis was ˜3.8 (i.e. −log₁₀[0.5¹²]).

Genomic DNA Sequencing

Genomic DNA of two patients, 21 family members and three independent controls was sequenced for mutations in exons 1 and 2 of Orai1 using the following oligonucleotide primers: Orai1ex1for1 5′ ACAACAACGCCCACTTCTTGGTGG (SEQ ID NO: 22) (exon 1); Orai1ex1rev1 5′ TGCTCACGTCCAGCACCTC (SEQ ID NO: 23) (exon 1); Orai1ex2for1 5′ TCTTGCTTTCTGTAGGGCTTTCTG (SEQ ID NO: 24) (exon 2); Orai1ex2rev1 5′ TCTCAAAGGAGCTGGAAGTGC (SEQ ID NO: 25) (exon 2). DNA was amplified using AmpliTaq Gold polymerase and separated on 1% agarose gels. PCR products were gel-purified and sequenced directly using the following primers: Orai1ex1for2 5′ AGCATGCAAAACAGCCCAGG (SEQ ID NO: 26) (exon 1); Orai1ex1rev2 5′ ACGGTTTCTCCCAGCTCTTC (SEQ ID NO: 27) (exon 1); Orai1ex2for2 5′ TGACAGGAGGAGAGCTAGG (SEQ ID NO: 28) (exon 2); Orai1ex2rev2 5′ AAGAGATCCTCCTGCCTTGG (SEQ ID NO: 29). Sequencing was done at the DF/HCC DNA Resource Core (DFCI) and DNA sequences analyzed using Xplorer Lite (dnaTools, Ft. Collins, Colo.).

Sequenom Analysis of HapMag_DNA

To exclude the possibility that the C>T point mutation at position 271 in the coding sequence of Orai1 (NM_(—)032790) is a SNP, we examined DNA from a panel of 270 individuals of diverse geographical origin assembled for the International HapMap project^(30,31). Genotyping was performed using a high-throughput primer extension method with detection by mass spectrometry (MALDI-TOF) on the Sequenom platform as previously described 53. A detailed description of this method can be found at http://www.hapmap.org/downloads/genotyping_protocols.html under “Sequenom platform”. 89% of samples were genotyped successfully and all were identified as CC homozygotes.

dsRNA Mediated Knockdown in Drosophila Cells

PCR fragments (size up to 600 bp) were used as templates for in vitro transcription reactions, followed by DNase I treatment to remove the template DNA. After purification, dsRNA (5 μg) was co-transfected together with the NFAT-GFP expression plasmid into S2R+ cells in 8-chamber slides (10 μg for 12 well plate). After 72 hrs of incubation, cells were treated with the Ca²⁺ influx inducers, 1 μM ionomycin or 1 μM thapsigargin for localization assays and were trypsinized for the measurement of [Ca²+]_(i) levels.

Genome-Wide RNAi Screen

The RNAi screen was performed essentially as described (Armknecht S. et al., 2005, Methods Enzymol 392, 55-73; Btros M. et al. 2004 Science 303, 832-835). The macrophage-hemocyte-like Drosophila cell line S2R+was stably transfected with the coding sequence for the NFAT1 (1-460)-GFP fusion protein subcloned into the expression plasmid pAc5.1 (Invitrogen). Transfection was achieved using Effectene (Qiagen) with a 19:1 ratio of the expression plasmid to pCoHygro (Invitrogen), which encodes a hygromycin resistance gene under the control of a constitutively active promoter. The cells were selected for 3-4 weeks with 300 μg/ml hygromycin, and stable clones were selected by visual inspection. 104 S2R⁺ cells stably expressing NFAT1 (1-460)-GFP were added onto each well of a 384 well plate containing 0.25 μg of dsRNAs (in 10 μl of serum-free medium) against Drosophila mRNAs and incubated for 1 h at 26° C. and incubated for 48-72 hrs at 26° C. to achieve RNAi. S2R⁺ cells were stimulated with 1 μM thapsigargin in Schneider medium containing 5 mM CaCl₂ at room temperature for 10 min, fixed and stained with DAPI. Coincident GFP and DAPI images were acquired by an automated camera from three different locations in each well, and scored by visual inspection. A total of fifty-eight 384-plates were analysed, containing a total of 21,884 wells into which individual dsRNAs had been arrayed. For this study, we note that the dsRNA amplicons for both dStim and dOrai had no predicted off-targets with exact matches of 19 nucleotides or greater.

Plasmids and Retroviral Transduction

Full-length cDNA for Orai1 (BC015369) was purchased from OpenBiosystems (Huntsville, Ala.) and subcloned into pENTR11 (“Gateway” system, Invitrogen, Carlsbad, Calif.) in frame with an N- or C-terminal terminal sequence encoding the myc epitope. Orai1 was then moved to the bicistronic retroviral expression vector pMSCV-CITE-eGFP-PGK-Puro (kind gift of Masatsugu Oh-hora), which allows for simultaneous expression of Orai1, GFP and a puromycin resistance gene. gp293 packaging cell lines were co-transfected with plasmids encoding Orai1, gag-pol and env to produce amphotropic, replication-incompetent retrovirus. Virus containing supernatant was collected for 24 h, filtered (0.45 microm, low protein binding) and concentrated by centrifugation at 6000×g for 16 h. T cells and fibroblasts were transduced by addition of viral supernatant for 4d and 1d, respectively. Transduction efficiency was evaluated by GFP expression using flow cytometry and myc-Orai1 expression using immunoblotting and immunocytochemistry. In some cases, transduced T cells were further selected with 1 μg/ml puromycin for 3 days.

Bioinformatic Prediction of Membrane Topoplogy

The hydropathy plot of Orai1 was generated using the Kyte-Doolittle algorithm²⁹. Membrane topology was further evaluated using the Phobius algorithm based on the hidden Markov model²⁶. Sequence alignment was performed using MegAlign (DNAStar, Madison, Wis.).

Confocal Imaging

Immunocytochemistry for Orai1 was done as described¹¹. Briefly, retrovirally transduced T cells and fibroblasts were fixed with 3% paraformaldehyde, left unpermebealized or permeabilized with wash buffer containing 0.5% NP-40, incubated with anti-myc antibodies (9E10) and Cy3-labeled secondary antibodies. Immunofluorescence was analyzed by confocal imaging using a Radiance 2000 Laser-scanning confocal system (Bio-Rad Laboratories) on a BX50BWI Olympus microscope using a 63× water immersion objective.

Single-Cell Ca²⁺ Imaging

T cells were loaded at 1×10⁶ cells/ml with 1 μM fura-2/AM (Molecular Probes) in loading medium (RPMI+10% FBS) for 30 min at 22-25° C., resuspended in loading medium and attached to poly-L-lysine-coated coverslips for 15 min. Fibroblasts were grown directly on UV-sterilized coverslips and loaded with 3 μM fura-2/AM for 45 min at 22-25° C. For [Ca²+]_(i) measurements, cells were mounted in a RC-20 closed-bath flow chamber (Warner Instrument Corp., Hamden, Conn.) and analyzed on an Axiovert S200 epifluorescence microscope (Zeiss) with OpenLab imaging software (Improvision). Cells were perfused in Ca²⁺-free Ringer solution and Ca²⁺ stores were passively depleted with 1 μM thapsigargin. Active depletion of stores was induced by incubation with 10 μg/ml anti-CD3 antibody (OKT3, eBioscience, San Diego, Calif.) for 10 min at 22-25° C. Fura-2 emission was detected at 510 nm with excitation at 340 and 380 nm and Fura-2 emission ratios (340/380) were calculated for each 5-s interval after subtraction of background. For each experiment, approximately 100 individual cells were analyzed for 340/380 ratios using Igor Pro (Wavemetrics, Lake Oswego, Oreg.) analysis software. [Ca²+]_(i) was estimated from the relation [Ca²⁺]_(i)=K*(R−R_(min))/(R_(max)−R). K*, R_(min), and R_(max) were measured in control human T cells in situ as previously described 54. Ca²⁺ influx rates were calculated from the maximal rate of rise in Ca²⁺ concentrations (d[Ca²⁺]_(i)/dt) after readdition of 0.2 mM extracellular Ca²⁺.

Ca²⁺ influx in S2R+ cells was measured by flow cytometry after detaching cells from the dish with trypsin (CellGro, Herndon, Va.). Cells were loaded with the Ca²⁺ indicator dyes Fluo4-AM and Fura-Red (2 μM each, Molecular Probes, Eugene, Oreg.) for 45 min at room temperature and then resuspended in loading medium (Schneider's medium+10% FCS). Immediately before the flow cytometric Ca²⁺ measurements, cells were resuspended in Ringer solution containing 2 mM Ca²⁺ and analyzed on a FACSCalibur (BD Biosciences, San Jose, Calif.). After 30 sec, thapsigargin (3 μM) in Ca²⁺ free Ringer to deplete intracellular Ca²⁺ stores, 4 mM Ca²⁺ Ringer solution was added and cellular Ca²⁺ levels were monitored for 300 sec. The ratio of Fluo-4 and Fura-Red emission was analyzed using FloJo software (Tree Star, Inc., Ashland, Oreg.).

Solutions and Chemicals

The standard extracellular Ringer's solution contained (in mM): 155 NaCl, 4.5 KCl, 20 CaCl₂, 1 MgCl₂, 10 D-glucose, and 5 Na-Hepes (pH 7.4). The standard divalent-free (DVF) Ringer's solutions contained (in mM): 155 Na, 10 HEDTA, 1 EDTA and 10 Hepes (pH 7.4). Charybdotoxin (CTX) was included in all external solution to block Kv1.3 channels to prevent contamination of I_(CRAC) recordings in DVF solutions. The standard internal solution contained (in mM): 150 Cs-aspartate, 8 MgCl₂, 8 BAPTA, and 10 Cs-Hepes (pH 7.2).

Thapsigargin (LC Biochemicals, Woburn, Mass.) was diluted from a 1 mM stock in DMSO, CTX (Sigma, St. Louis, Mo.) was diluted 1:1000 from 50 μM stock solution in water. 2-aminoethyoxydiphenylborate (2-APB, Sigma) was diluted from stock solutions in DMSO. The drugs were diluted to the concentrations indicated in the legends and applied to the cells using a multi-barrel local perfusion pipette with a common delivery port. The time for 90% solution exchange was measured to be <1 s, based on the rate at which the K⁺ current reversal potential changed when the external [K⁺] was switched from 2 mM to 150 mM.

Patch-Clamp Measurements

Patch-clamp experiments were conducted in the standard whole-cell recording configuration at 22-25° C. using an Axopatch 200 amplifier (Axon Instruments, Foster City, Calif.) interfaced to an ITC-16 input/output board (Instrutech, Port Washington, N.Y.) and a Macintosh G3 computer. Recording electrodes were pulled from 100-μl pipettes, coated with Sylgard, and fire-polished to a final resistance of 2-5 MΩ. Stimulation and data acquisition and analysis were performed using in-house routines developed on the Igor Pro platform (Wavemetrics, Lake Oswego, Oreg.). The holding potential was +30 mV unless otherwise indicated. Voltage stimuli usually consisted of a 100-ms step to −100 mV followed by a 100-ms ramp from −100 to +100 mV, applied every 1.3 s. Currents were filtered at 2 kHz with a 4-pole Bessel filter and sampled at 5 kHz. Data are corrected for the liquid junction potential of the pipette solution relative to Ringer's in the bath (−10 mV) and for the bath DVF solution relative to Ringer's in the bath-ground agar bridge (+5 mV). For noise analysis, 200-ms sweeps were acquired at the rate of 3 Hz at a holding potential of −100 mV, digitized at 5 kHz, and low-pass filtered using the Axopatch 200 amplifier's internal Bessel filter at 2 kHz. The mean and variance were calculated from 100-ms segments of the digitized data.

Data Analysis

Unless noted otherwise, all data were corrected for leak currents collected either with 2 μM La³⁺ or with traces collected prior to I_(CRAC) induction during passive dialysis with BAPTA. Permeability ratios (P_(Cs)/P_(Na)) was calculated from the biionic reversal potential using the equation:

$\frac{P_{Cs}}{P_{Na}} = {\left( \frac{\lbrack{Na}\rbrack_{o}}{\lbrack{Cs}\rbrack_{i}} \right)^{(\frac{E_{rev}F}{RT})}}$

where R, T, and F have their usual meanings and E_(rev) is the reversal potential.

Introduction

Ca²⁺ is an essential second messenger in almost all cell types. In particular, sustained Ca²⁺ influx across the plasma membrane is crucial for lymphocyte activation and the adaptive immune response¹. Antigen recognition by the surface antigen receptors of T and B lymphocytes triggers an elaborate signal transduction cascade, involving the activation of multiple tyrosine kinases and the assembly of large scaffolded complexes containing diverse adapters and signaling proteins. An early biochemical consequence is the activation of PLCγ, which releases Ca²⁺ from the endoplasmic reticulum (ER) by generating IP₃; the resulting decrease in lumenal ER Ca²⁺ opens a class of “store-operated” Ca²⁺ channels with very specific electro-physiological characteristics, which have been termed Ca²⁺ release-activated Ca²⁺ (CRAC) channels¹⁻³. CRAC channel opening results in sustained influx of Ca²⁺ ions across the plasma membrane, promoting a sustained elevation of intracellular free Ca²⁺ ([Ca²+]_(I)) levels and activating diverse Ca²⁺/calmodulin-dependent enzymes including the protein phosphatase calcineurin; an ultimate consequence is the activation of Ca²⁺-dependent transcriptional pathways required for proliferation and effector immune function^(4,5). One of the major Ca²⁺-regulated transcription factors is NFAT, a family of heavily-phosphorylated proteins that resides in the cytoplasm of resting cells . Sustained Ca²⁺ influx results in the dephosphorylation of NFAT by calcineurin and promotes its translocation to the nucleus, where it turns on the expression of a large number of activation-associated genes^(4,6).

A great deal of pharmacological, electrophysiological, and genetic evidence supports the notion that CRAC channels are the principal pathway for Ca²⁺ influx in both developing and mature T cells, thus orchestrating essentially all aspects of lymphocyte development and function^(1,7). Analysis of two families of patients with hereditary severe combined immune deficiency (SCID), who presented as infants with a marked propensity to bacterial and viral infections, revealed that the primary defect is lack of store-operated Ca²⁺ entry in the patients' lymphocytes⁸⁻¹⁰. Detailed analysis of T cell lines derived from one family of patients revealed severe impairment of NFAT dephosphorylation, nuclear translocation and activation of NFAT-dependent genes, secondary to a correspondingly severe impairment of store-operated Ca²⁺ influx in cells activated through the T cell receptor or treated with thapsigargin, an inhibitor of the SERCA Ca²⁺ pump¹⁰. Electrophysiological analysis of the patients' T cells confirmed an almost complete absence of CRAC channel function¹¹. Together these data highlight the crucial importance of CRAC channels and store-operated Ca²⁺ entry for lymphocyte activation and immune defense.

Although the pharmacological and electrophysiological properties of the CRAC channel have been described in some detail^(1,12,13), its molecular identity has remained elusive to date. The key biophysical hallmarks of the channel include high selectivity for Ca²⁺ over monovalent cations, low single-channel conductance (<1 pS), an inwardly rectifying I-V relationship, a lack of significant voltage-dependent gating, rapid inactivation by intracellular Ca²⁺, extracellular blockade by submicromolar La³⁺, and modulation of channel properties by 2-APB^(1,13,14). Several candidate genes belonging to the TRP family of ion channels have been proposed to encode the CRAC channel, including TRPC1¹⁵, TRPC3¹⁶, and TRPV6^(17,18), as well as voltage-gated Ca²⁺ (Cav) channels 1920. However, evidence that TRPs are store-dependent following heterologous expression in several cell lines is inconsistent^(21,22), and none of the candidates exhibit all of the biophysical properties of the CRAC channel. Previous sequence analyses and complementation studies in the SCID patients' cells had failed to establish a role for several TRP family members including TRPC3, TRPV5 and TRPV6 in the defect in CRAC channel function¹¹. More recently, the type I membrane proteins STIM1 and STIM2 were shown to be essential for store-operated Ca²⁺ entry and CRAC channel function 23,24. STIM1 has been suggested to “sense” the filling state of the ER Ca²⁺ stores via its EF hand domain, thus coupling store depletion to the opening of CRAC channels. However neither STIM1 nor STIM2 were mutated in the SCID patients, and expression of STIM1 in SCID T cells did not result in complementation of the Ca²⁺ entry defect¹¹.

Here we describe the identification of a novel protein crucial for store-operated Ca²⁺ entry and CRAC channel function. The protein, here termed Orai1, was identified using two unbiased genetic approaches: a modified linkage analysis to identify the gene mutated in the SCID patients, and a genome-wide RNAi screen in Drosophila to identify regulators of store-operated Ca²⁺ entry and NFAT nuclear import. The combination of these two approaches pinpointed a single candidate gene. We show that RNAi-mediated depletion of Drosophila Orai abrogates store-operated Ca²⁺ entry as effectively as RNAi against Drosophila Stim. We further show that a point mutation in Orai1 is responsible for the Ca²⁺ influx defect in the SCID patients, and that complementation of SCID T cells and fibroblasts with wild type Orai1 reconstitutes store-operated Ca²⁺ influx and CRAC channel current (I_(CRAC)). The pharmacological and electrophysiological properties of the reconstituted currents are indistinguishable from those of endogenous I_(CRAC) in control T cells. The primary sequence of Orai1 predicts four transmembrane domains, and immunocytochemistry of epitope-tagged Orai1 shows that the protein is localized at or near the plasma membrane.

Results Phenotypic Identification of Heterozygous Disease Carriers

The two SCID patients were born to consanguineous parents, suggesting an autosomal recessive mode of inheritance as neither the parents of the SCID patients nor any other members of the SCID patients' family showed clinical symptoms of immunodeficiency (FIG. 1A). Furthermore, T cells derived from the parents of the SCID patients showed almost normal store-operated Ca²⁺ entry in the presence of 2 mM extracellular Ca²⁺¹⁰. To unmask a potential defect in Ca²⁺ entry in the parental T cells, we measured the initial rate of Ca²⁺ influx (here defined as the initial rate of change of intracellular free Ca²⁺ concentration, d[Ca²⁺]_(i)/dt) after thapsigargin-mediated store depletion, but decreased the driving force for Ca²⁺ entry by reducing the extracellular Ca²⁺ concentration from 2 mM to 0.2-0.5 mM CaCl₂. Under these conditions, peak Ca²⁺ levels and Ca²⁺ influx rates in T cells from both parents were ˜50% or less of those observed in wild-type control T cells (FIG. 1B). We hypothesized that this finding reflected a potential gene-dosage effect, resulting from the fact that the parents were heterozygous carriers of the causal mutation in the SCID patients.

We used this assay to identify other potential heterozygous carriers of such a mutation in the more extended pedigree. Blood samples were obtained from 19 additional family members (FIG. 1A), T cell lines were generated, and Ca²⁺ entry phenotype was evaluated by phenotypic analysis in vitro. Thirteen family members consistently showed reduced peak Ca²⁺ influx and decreased initial rate of Ca²⁺ influx, compared to T cells from 8 other family members and unrelated controls (FIG. 1C). An arbitrary cutoff of Ca²⁺ influx rate below 2 nM/s was used to distinguish potential heterozygous disease carriers from unaffected (homozygous wild-type) individuals (FIG. 1C). With this cutoff, the distribution of putative heterozygous carriers within the family appears fully compatible with an autosomal dominant mode of inheritance (FIG. 1A).

Linkage Mapping by Genome-Wide SNP Array Screen

Genomic DNA from the 23 members of the SCID family was used for genotyping using genome-wide SNP arrays. SNP data were evaluated using two independent linkage analyses. The first analysis assumed an autosomal recessive mode of inheritance based on the clinical phenotype, and DNA from the two patients, their parents, their unaffected brother and their grandparents was analysed (Pedigree A, indicated by the grey shaded area in FIG. 1A). In contrast, the second analysis utilized the remainder of the pedigree in a completely independent analysis. Here, an autosomal dominant mode of inheritance was assumed, based on our ability to identify heterozygous carriers of the disease mutation by phenotypic analysis in vitro (Pedigree B, indicated by the green box in FIG. 1A). Importantly, the first analysis (standard homozygosity mapping) was performed without consideration of the heterozygous phenotype status of individuals, and the second (dominant inheritance) was performed on the large pedigree as two unrelated halves (the relatives of parent 35 and 36 being treated independently) such that the results of these two analyses are fully independent. Thus we can consider the analyses of these two runs to emerge from three independent pedigrees (one homozygosity mapping run and two unrelated dominant pedigrees) and can simply add the parametric LOD scores from these to acquire a statistically robust combined LOD score (see Materials and Methods).

Parametric linkage analysis for a recessive trait (Pedigree A) identified six regions on six chromosomes with LOD scores of 1.5-1.9—while one of these is almost certain to harbor the gene, it is fully expected that this maximum LOD score would be achieved several times by chance and thus the homozygosity mapping is not sufficient alone to map this gene. Satisfyingly, the dominant analysis identified a unique region on chromosome 12q24, clearly overlapping with one of the 6 regions identified in the homozygosity mapping analysis, with a LOD score of ˜3.8. The combination of these two linkage analyses defines an overlapping ˜9.8 Mb candidate region with a highly significant cumulative LOD score of 5.7, representing odds of ˜500,000:1 in favor of linkage—overwhelmingly likely to contain the true gene. This region is located between SNP_A-1514003 and SNP_A-1510776 (115.49 Mb-125.27 Mb). In support of this conclusion, no other region in the genome had a cumulative LOD score exceeding zero. Because incorrect assignment of heterozygous disease carrier status based on phenotypic analysis would decrease overall LOD scores rather than yielding false positives of this magnitude, our novel combination of recessive and dominant analyses successfully identifies a genomic region with a very high probability of linkage to the mutant gene.

Genomic sequencing of six known genes in this region with a potential role in Ca²⁺ signaling or Ca²⁺ binding (PLA2G1B, CABP1, P2RX7, P2RX4, CAMKK2, PITPNM2) did not reveal any mutations in exons or immediately adjacent genomic regions. It did however allow us to narrow down the candidate homozygous region from ˜9.8 Mb to ˜6.5 Mb, on the basis of several SNPs in PITPNM2 for which the patients were heterozygous. The ˜6.5 Mb interval contains ˜74 genes, of which 16 were annotated as “hypothetical proteins” or potential gene loci (Human genome assembly, NCBI build 35.1). Of these, 2 were predicted to contain transmembrane domains (KIAA0152 and FLJ14466) using TMHMM and Phobius algorithms^(25,26).

A Genome-Wide RNAi Screen in Drosophila Identifies olf186F (dOrai) as a Novel Regulator of Store-Operated Ca²⁺ Entry

In parallel with the positional cloning effort, we conducted a genome-wide RNAi screen for NFAT regulators in Drosophila, as an independent method of identifying components of the CRAC channel and the signalling pathway leading to CRAC activation. Drosophila S2R+ cells, stably-expressing an NFAT-GFP fusion protein, were incubated for 3 days with arrayed dsRNAs against each of 21,000 Drosophila genes to achieve knockdown of gene expression. The cells were then stimulated for 10 min with thapsigargin to deplete Ca²⁺ stores, thus activating store-operated Ca²⁺ entry and nuclear translocation of NFAT-GFP. The cells were then fixed, wells containing the cells were photographed robotically, and the subcellular distribution of NFAT-GFP was assessed by visual inspection. Among the positive candidates whose depletion interfered with NFAT nuclear translocation were several expected regulators of the Ca⁺/calcineurin/NFAT signalling pathway, including Calcineurin B (CanB), Calcineurin A (CanA-14F) and Drosophila Stim^(24,27).

One positive candidate, olf186F, was notable because the gene encoding one of its three human homologues was located within the 6.5 Mb homozygous genomic region linked to the SCID mutation at 12q24 (hypothetical protein FLJ14466, NM_(—)032790, NP_(—)116179). For reasons discussed below, olf186F and its human homologue at 12q24 have been designated Drosophila Orai (dOrai) and human Orai1 respectively; the other two human homologues, C7Orf19 located on chromosome 7 and MGC13024 located on chromosome 16, have been designated Orai2 and Orai3 (FIG. 3A). In Drosophila S2R+ cells, RNAi-mediated depletion of either dStim or dOrai blocked nuclear translocation and dephosphorylation of NFAT-GFP (FIG. 2B). Likewise, knockdown of either dSTIM or dOrai completely inhibited thapsigargin-induced Ca²⁺ influx in S2R+ cells (FIG. 2B). These data confirm previous reports that dSTIM and human STIM1 are essential for store-operated Ca²⁺ entry and CRAC channel activation in Drosophila and mammalian cells^(23,24,28), and identify dOrai as a second novel regulator of store-operated Ca²⁺ entry in Drosophila cells.

Orai1 is Mutated in the SCID Patients

Since our data implicated dOrai as a second novel regulator of store-operated Ca²⁺ entry (FIG. 2), and since the gene for human Orai1 was located in the 12q24 region that is homozygous in the SCID patients, we asked whether the SCID defect was associated with a mutation in human Orai1 (FIG. 3). By sequencing genomic DNA from the 23 individuals (patients and their relatives) shown in FIG. 1A, we found that both SCID patients were homozygous for a missense mutation in exon 1 of Orai1. The mutation at position 271 of the coding sequence of Orai1 (position 444 of NM_(—)032790), a C>T transition, leads to substitution of tryptophan for a highly-conserved arginine residue at position 91 (R91W) of the protein (NP_(—)116179, FIG. 3B). The mutated residue is located at the beginning of the first of four potential transmembrane segments in Orai1, predicted by calculating the hydrophobicity of Orai1 using the Kyte-Doolittle method²⁹ (FIGS. 3B, 3C). All 13 phenotypically predicted heterozygous disease carriers (FIG. 1) were genotypically heterozygous for the mutation (C/T), while healthy controls and unaffected family members were homozygous for the wild-type allele (C/C). The mutation at this position is not an annotated SNP (dbSNP Build 124), rendering it unlikely this is simply a common polymorphism. To confirm this hypothesis, we typed this polymorphism in the entire HapMap panel (270 individuals in total from Utah, Ibadan (Nigeria), Tokyo and Beijing) and did not find a single copy of the putatively causal “T” allele in this panel (Materials and Methods, and data not shown)^(30,31). These data demonstrate unequivocally that the C>T transition is not a common sequence variant in the general population; thus the mutation is likely to have occurred spontaneously in the ancestors of the SCID patients and is strongly associated with disease.

Expression of Orai1 Restores Store-Operated Ca²⁺ Influx in the SCID T Cells

We asked whether Orai1 would complement the Ca²⁺ influx defect in the SCID T cells (and fibroblasts) by expressing N- and C-terminally epitope-tagged versions of wild type and mutant Orai1 in T cells and fibroblasts from the SCID patients. Retroviral expression of Myc-Orai1^(WT) in SCID T cells or fibroblasts using a bicistronic IRES-GFP vector restored Ca²⁺ influx in response to thapsigargin treatment in GFP-positive but not GFP-negative cells, whereas retroviral expression of mutant R91>W Orai1 (Myc-Orai1^(R>W)) did not restore Ca²⁺ influx. The inability of Myc-Orai1^(R>W) to restore Ca²⁺ influx in the SCID T cells and fibroblasts was not due to aberrant expression of Myc-Orai1^(R>W) compared to Myc-Orai1^(WT), because mutant and wild-type proteins are present at equivalent levels and appear to be similarly localized at or near the plasma membrane as judged by immunoblotting (data not shown) and immunocytochemistry. We were unable to stain non-permeabilized cells with the anti-myc antibody, consistent with a topology in which both the N- and C-termini are cytoplasmically oriented and so inaccessible to the antibody (FIG. 3C).

Notably, Ca²⁺ influx in SCID T cells (and fibroblasts) reconstituted with Myc-Orai1^(WT) did not occur in unstimulated T cells (or fibroblasts) when 2-20 mM extracellular Ca²⁺ was present but was only observed after store-depletion with thapsigargin (FIG. 5A-5D). This is an important finding because it demonstrates that restoration of Ca²⁺ influx in Orai1-expressing cells is dependent on store depletion, a defining feature of store-operated Ca²⁺ entry through CRAC channels, and is not due to expression or activation of constitutively-open Ca²⁺ channels. Myc-Ora1^(WT) also restored store-operated Ca²⁺ entry in SCID T cells in response to TCR crosslinking. The pharmacological characteristics of thapsigargin- and TCR-induced Ca²⁺ entry in SCID T cells and fibroblasts complemented with Orai1 were exactly those expected for Ca²⁺ influx through CRAC channels^(12,32). Treatment with 75 μM 2-APB or 2 μM La³⁺ inhibited Ca²⁺ influx (FIGS. 5A,5C,5D), whereas treatment with a low dose of 2-APB (3 μM) caused a distinct further increase in [Ca²+]_(i) (FIG. 5B), although the increase in the Orai1^(WT) expressing SCID T cells was slightly lower than that in control T cells (˜15% vs. ˜23%). Taken together, these results show clearly that Orai1 is the gene responsible for the Ca²⁺ influx defect in the SCID patients' T cells and fibroblasts.

Expression of Orai1 Restores I_(CRAC) in the SCID T Cells

The recovery of Ca²⁺ influx seen in the previous experiments could reflect reconstitution of active CRAC channels in the patients' cells, or could arise from expression (or activation) of store-operated, Ca²⁺ permeable ion channels distinct from CRAC. To distinguish between these possibilities, we characterized in detail the current arising from store-depletion in the SCID cells reconstituted with wild type or mutant (R91W) Orai1, using the whole-cell patch-clamp configuration. SCID T cells were retrovirally transduced with Orai1 in a bicistronic IRES-GFP vector, and cells expressing Orai1 were identified by GFP fluorescence as described above. In the experiments shown here, store depletion was accomplished either by including 8 mM BAPTA in the patch pipette or by treatment with thapsigargin.

In SCID cells reconstituted with wild type Orai1, inclusion of 8 mM BAPTA in the patch pipette caused the slow development of an inward current in 20 mM Ca²⁺ _(o), following whole-cell break-in, reminiscent of the development of I_(CRAC) in response to store depletion (FIG. 4A)^(2,3). By contrast, SCID T cells expressing the R91W mutant of Orai1 failed to manifest any inward Ca²⁺ currents following store depletion either with BAPTA (FIG. 4C) or with thapsigargin (data not shown), as expected from the inability of this mutant protein to reconstitute store-operated Ca²⁺ entry. The current observed in Orai1-reconstituted SCID T cells displayed many key hallmarks of the I_(CRAC) ^(11,33,34). First, when a divalent-free (DVF) solution lacking Ca²⁺ and Mg²⁺, in which the only current carrier is Na⁺, was applied after full development of the current in 20 mM Ca²⁺ _(o), an inward Na⁺ current was observed that was initially much larger than the Ca²⁺ current but that declined over tens of seconds (FIG. 4A). This decline of the Na⁺ current, known as depotentiation, is characteristic of CRAC channels in Jurkat T cells, RBL cells and human T cell lines^(11,33,34). Second, both the Ca²⁺ and Na⁺ currents showed an inwardly rectifying current-voltage (I-V) relationship (FIG. 4B). The reversal potential of the inward current in 20 mM Ca²⁺ was >+90 mV, consistent with the known high selectivity of CRAC channels for Ca²⁺, whereas the reversal potential in divalent-free solution was 49±2 mV (n=4 cells), indicating that the channels are only weakly permeable to the Cs⁺ ions in the patch pipette (P_(Cs)/P_(Na)=0.14) and consistent with the selectivity of CRAC channels for monovalent ions^(33,35). Third, the noise characteristics of the Orai1 complemented current were consistent with those of CRAC channels in wild-type T cells(FIG. 4D)³³. During depotentiation of the Na⁺ current, variance declined linearly with mean current with an average slope of 29±4 fA (n=4 cells), providing a lower limit estimate of the unitary current similar to that of previous measurements of I_(CRAC). Furthermore, the Ca²⁺ current resulting from complementation with Orai1 exhibited fast inactivation in 20 mM Ca²⁺ _(o) (FIG. 4E); the extent and time course of inactivation was similar to that previously reported for CRAC channels in Jurkat T cells (current inactivates by 54±5% at −100 mV within 200 ms; τ_(fast): 9±2 ms; τ_(slow): 84±12 ms)³⁶. And lastly, the pharmacological hallmarks of the reconstituted current included complete block by 2 μM La³⁺ (FIG. 4F), inhibition by high doses of 2-APB (FIG. 4G) and potentiation by low doses of 2-APB (FIG. 4G); moreover the block observed with high doses of 2-APB was accompanied by the loss of fast inactivation³². The discrepancy between full complementation of CRAC currents by expression of Orai1 (FIG. 4H) and the partial complementation of Ca²⁺ influx observed by Ca²⁺ imaging may be explained by the fact that for measurements of I_(CRAC), we selected T cells with high GFP/Orai1 levels, whereas for the single-cell Ca²⁺ imaging, we averaged responses of all GFP/Orai1-positive cells (both bright and dim).

In summary, reconstitution of SCID T cells with Orai1 restores not only store-operated Ca²⁺ entry but also a current that is identical to I_(CRAC) with regard to store dependence, ion selectivity and unitary conductance, gating properties, and pharmacological profile. Thus, we conclude that Orai1 is essential for CRAC channel function in T cells. The pore properties and pharmacological characteristics of the channel observed in SCID T cells complemented with Orai1 are indistinguishable from those of bonafide CRAC channels.

Discussion

Here we identify Orai1 as an evolutionarily-conserved component of store-operated Ca²⁺ entry and an essential contributor to I_(CRAC). We show that a point mutation in Orai1 is responsible for the genetic defect in store-operated Ca²⁺ entry and I_(CRAC) function in two patients with a rare form of severe combined immune deficiency (SCID)^(10,11). Identification of Orai1 as the defective gene was accomplished through the synergistic combination of two independent genetic analyses, both involving unbiased genome-wide screens.

Our first screen employed genome-wide SNP analysis to identify the chromosomal region linked to the SCID disease. Because only two diseased individuals exist, the theoretically-attainable LOD score from traditional linkage analysis is 1.9, significantly below the 3.0 value necessary to establish linkage. Indeed, analysis of a small pedigree including the two SCID patients, their parents and their grandparents identified 6 regions on 6 separate chromosomes with maximum LOD scores of 1.9 (Pedigree A). To extend the amount of genetic information available, we devised a method of identifying heterozygous carriers of the mutant allele. This was accomplished through a simple modification of our in vitro method of measuring store-operated Ca²⁺ influx, in which the driving force for Ca²⁺ entry was decreased by reducing the extracellular Ca²⁺ concentration. When this assay was applied to T cell lines derived from 21 additional family members of the SCID patients (Pedigree B), 13 members showed a significantly reduced initial rate of Ca²⁺ influx, which we interpret as reflecting a gene-dosage effect consistent with heterozygosity for the mutant allele. A second, completely independent linkage analysis, in which the haplotype of these 13 putatively heterozygous individuals was compared to that of the remaining 8 homozygous healthy family members, yielded experimental LOD scores that identified a unique region on 12q24 with a LOD score of 3.8. This region overlapped with one of the regions identified by linkage analysis of Pedigree A. Because the individuals used for each analysis and the phenotypes used to classify them were distinct, allele sharing and thus linkage results were completely independent in these analyses; hence we could combine LOD scores from the two analyses to obtain an unbiased cumulative and highly significant LOD score of ˜5.7 for an ˜9.8 Md region at 12q24. In principle, this novel and powerful combination of linkage mapping approaches may be applied to elucidate the genetic causes of other rare autosomal-recessive diseases, even if only a very few diseased individuals are available and conventional homozygosity mapping fails to establish linkage. Prerequisites are that other family members are available and that mutation of one allele can be detected as a quantifiable trait in vitro.

In the hope of rapidly identifying a gene in the 12q24 region that was involved in store-operated Ca²⁺ entry, we conducted a parallel genome-wide RNAi screen in Drosophila, taking advantage of the fact that Drosophila S2R cells contain a store-operated Ca²⁺ channel with characteristics very similar to CRAC³⁷. Rather than focusing solely on Ca²⁺ entry, we designed the screen to identify evolutionarily-conserved regulators of the Ca²⁺-regulated transcription factor NFAT; although Ca²⁺-regulated NFAT proteins are not themselves represented in Drosophila, there is strong evolutionary conservation of the pathways which regulate its nuclear-cytoplasmic shuttling, through effects on Ca²⁺ homeostasis, store-operated Ca²⁺ entry, calcineurin activity and kinase-phosphatase balance²⁷. The screen was used to identify candidates whose RNAi-mediated depletion interfered with nuclear localization of an NFAT-GFP fusion protein in response to stimulation with thapsigargin. Among the positive candidates was olf186F (here renamed Drosophila Orai), which has three human homologues, FLJ14466, C7Orf19 and MGC13024. Since these are novel proteins without known function, we named them Orai1-3, respectively. In Greek mythology, the Orai are the keepers of the gates of heaven: Eunomia (Order or Harmony), Dike (Justice) and Eirene (Peace)³⁸⁻⁴⁰; in Japan, Orai is in part derived from the sound of “all right” in English and also refers to comings and goings, communication, streets and traffic in Japanese. In a satisfying validation of our dual strategy, the gene encoding Orai1 (hypothetical protein FLJ14466) is located on chromosome 12q24, exactly the region identified by our SNP analysis as linked genetically to the SCID syndrome. DNA sequencing rapidly revealed the genetic basis for the SCID defect as a point mutation (C>T) in exon 1 of Orai1, which resulted in an arginine to tryptophan substitution at residue 91. This mutation is not a known polymorphism, as confirmed by sequencing DNA from 270 individuals of mixed ethnic backgrounds assembled for the international HapMap project³¹. This number of samples is sufficient to find almost all haplotypes with frequencies of 5% or higher. Although there is a small chance that the C>T mutation is a SNP confined to a small ethnic population not represented in the HapMap panel, this possibility can be ruled out with reasonable certainty based on the fact that complementation with Orai1 restores store-operated Ca²⁺ entry and I_(CRAC) in SCID patient cells. Furthermore, arginine 91 which is mutated in the SCID patients is located in a putative transmembrane region that is highly conserved across species (FIG. 3A), highlighting its potential importance in the function of Orai1.

The characteristics of Ca²⁺ influx and Ca²⁺ current in Orai1-complemented SCID T cells were indistinguishable from those observed in control T cells. In particular, both processes were strictly regulated by store depletion, and the electrophysiological and pharmacological properties of the restored current were fully consistent with those of I_(CRAC). These properties include: an extremely high selectivity for Ca²⁺ over monovalent cations, inwardly rectifying I-V relation, depotentiation under divalent-free conditions, current noise characteristics, rapid Ca²⁺-dependent inactivation, blockade by low micromolar La³⁺ and positive and negative modulation by 2-APB. We therefore conclude that Orai1 reconstituted I_(CRAC) in the SCID patients' T cells, and thus that the C>T transition and resulting R91W mutation in the Orai1 coding region and protein are responsible for the SCID defect. While its specific role has not yet been determined, the available data are consistent with the possibility that Orai1 encodes a channel subunit or a closely-associated channel regulator in the plasma membrane. First, the hydropathy profile of Orai1 predicts a membrane protein with three, or potentially four, hydrophobic membrane domains (FIG. 3B). Second, immunocytochemistry of myc-tagged Orai1 is consistent with localization at the plasma membrane under resting conditions; this distribution differs from that of STIM1, which is predominantly located in the ER where it is thought to sense Ca²⁺ store depletion via its luminal EF hand domain (Feske 2005, Liou 2005, Ref). Notably, both N- and C-terminal epitope tags on Orai1 are inaccessible to antibody staining in non-permeabilised cells; this finding is consistent with the prediction of four transmembrane domains and predicts a topology compatible with a channel subunit, in which both N- and C-termini are cytoplasmically oriented (FIG. 3C). Further studies will be necessary to determine whether Orai1 is part of the CRAC channel itself, or whether it encodes a regulator of the channel.

Orai1 is widely expressed at the mRNA level, potentially explaining our previous observations that not only T cells but also B cells and fibroblasts from the SCID patients show a substantial defect in store-operated Ca²⁺ entry. Surprisingly, however, the clinical phenotype of the SCID patients is predominantly one of immunodeficiency, associated in the single surviving patient with ectodermal dysplasia and anhydrosis (EDA) and a mild, congenital, non-progressive myopathy. EDA is characterized by defective tooth enamel and hair follicle function, and complete absence of sweat glands, and many previous studies have linked it to hypoactivation of NF-κB^(41,45). Ca²⁺ mobilization is thought to contribute to NFκB activation in T cells and other cell types under certain conditions of stimulation⁴⁶, thus the EDA syndrome may well reflect defective NFκB activation, either during development or acutely in specific cell types. In contrast the myopathy could potentially be a direct consequence of defective NFAT activation, given that NFAT has a major role in certain aspects of skeletal muscle development and function (reviewed in^(7,47)).

In conclusion, our studies establish a critical role for Orai1 in T cell function and the in vivo immune response. A single point mutation in Orai1, a novel protein conserved from C. elegans to humans, disrupts store-operated Ca²⁺ entry and CRAC channel function in patients with an inherited immune deficiency. Future studies will address the relation between Orai and Stim proteins and the mechanism by which store depletion couples to CRAC channel opening.

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Example 2 A Genome-wide Drosophila RNAi Screen Identifies DYRK as a Novel Regulator of NFAT Materials and Methods The Genome-Wide Primary Screen

Methods were adapted from refs^(12,13). 10⁴S2R⁺ cells were added into each well containing 0.25 μg of dsRNAs in 10 μl of serum-free medium and incubated for 1 h at 26° C. The cells were then transiently transfected with NFAT1 (1-460)-GFP expression plasmid^(9,17) (10 ng) in Schneider's medium (Invitrogen) (30 μl). After incubation for 48-72 hrs at 26° C., the cells were fixed and stained with DAPI, and the coincident GFP and DAPI images were acquired by an automated camera from three different locations in each well. A total of fifty-eight 384-plates were analysed, containing a total of 21,884 wells into which individual dsRNAs had been arrayed.

Control wells (no dsRNA, dsRNA against GFP, and dsRNA against a gene (thread—anti-apoptotic) causing cell death) were present on each plate and served as an internal control for knockdown efficiency of each plate. All three photographs of GFP fluorescence in each assay well were manually scored using MetaMorph 6.1 Software (Universal Imaging Corporation). To identify even weak effectors of NFAT localization non-stringent criteria were used in the primary screen, such that wells were scored positive even if only one cell in each of three fields showed complete nuclear localization of NFAT-GFP. Since the RNAi library was constructed before the Drosophila genome was completely annotated, 39 of the 738 positives did not correspond to known genes and were eliminated. Another 37 candidates were eliminated because the dsRNAs used to identify them had more than 10 predicted “off-targets” with exact matches of 21 nucleotides (nt) (see Bioinformatics and Classification below).

The Confirmatory Screen

The confirmatory screening on the 699 potentially positive candidates from the primary screen was performed essentially as described for the primary screen, except that S2R⁺ cells stably transfected with NFAT1(1-460)-GFP were used, and candidates were tested for whether their depletion altered NFAT subcellular localization in both resting and stimulated S2R⁺ cells. Wells in which all cells contained cytoplasmic NFAT-GFP got the lowest score (0) while wells with >90% of the cells showing nuclear NFAT-GFP scored the highest (3). The summed scores from all three experiments are presented in Table 1. Note that the highest possible score is 9, but because we scored conservatively in the confirmatory screen, the highest actual score obtained by any candidate is 6. All candidates were also tested for whether they prevented NFAT nuclear localization in cells treated with thapsigargin (1 μM, 30 min); only Drosophila STIM (dSTIM) scored positive in this assay.

To generate the stably-expressing cell line, the coding sequence for the NFAT1(1-460)-GFP fusion protein was subcloned into the expression plasmid pAc5.1 (Invitrogen), and the macrophage-hemocyte-like Drosophila cell line S2R⁺ was transfected in a 6-well format using Effectene (Qiagen) with a 19:1 ratio of the expression plasmid to pCoHygro (Invitrogen), which encodes a hygromycin resistance gene under the control of a constitutively active promoter. The cells were selected for 3-4 weeks with 300 μg/ml hygromycin, and stable clones were selected by visual inspection.

Bioinformatics and Classification

Scores were consolidated and formatted for submission to the DRSC (Drosophila RNAi Screening Center at Harvard Medical School), which then provided the identity of the genes assayed (FlyBase identifier; Drosophila gene name, where known; some Gene Ontology (GO) identifiers; and some human homologues). Gene Ontology (GO) annotation was retrieved in two ways. First, we employed Ensembl's EnsMart tool using the FlyBase identifier for each gene to get the GO description. Second, we used the GO identifiers provided by the screening center to get descriptions from the “GO terms and IDs” file from the Gene Ontology Consortium. Functional categories of genes were constructed by keyword searches of the positives followed by manual curation. Positive genes were also examined for involvement in common pathways using tools such as those at the KEGG Pathway Database.

For each candidate that was positive in the primary screen, the number of off-targets was determined using the off-target sequence search tool on the DRSC website (http://www.flyrnai.org/RNAi_primer_design.html). This bioinformatic tool is based on an algorithm similar to that in ref³⁷ except that it does not have a built-in primer design component (Flockhart et al., submitted). Amplicon (dsRNA) sequences are searched for predicted off-targets by considering all possible fragments, of length 16-50 bp with a default value of 21 bp, that perfectly match sequences in fly transcripts in release 4.0. Ideally, only 1 match corresponding to the targeted mRNA should be found, but some amplicons have matches with other mRNAs which are not the intended target. For the genes in Table 1, a default length of 21 nt was used to compute the number of off-targets for each positive candidate, and candidates with >10 off-targets were eliminated. For the genes in Table II (calcineurin) and III (candidates used for additional experiments), shorter fragments of 19 nt and 20 nt were considered as well. The identity of off-targets was determined using BLASTN against Drosophila NCBI RefSeq database. Mammalian orthologues of Drosophila melanogaster proteins in Table I were retrieved from the NCBI Homologene database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene). The human homologues of the fly kinases were obtained by reciprocal blast method using BLASTP; Altschul, et al. 1990, J. Mol. Biol. 215:403-410), as described^(38,39). Phylogenetic analysis was performed using TCoffee⁴⁰, and the reliability of the ortholog assignments was assessed with the bootstrap method implemented in Orthostrapper⁴¹.

DsRNA Mediated Knockdown in Drosophila Cells

PCR fragments (size up to 600 bp) were used as templates for in vitro transcription reactions, followed by DNase I treatment to remove the template DNA. After purification, dsRNA (5 μg) was co-transfected together with the NFAT-GFP expression plasmid into S2R⁺ cells in 8-chamber slides (10 μg for 12 well plate). After 72 hrs of incubation, cells were left untreated or were treated with the Ca²⁺ influx inducers, 1 μM ionomycin or 1 μM thapsigargin for localization assays and were trypsinized for the measurement of [Ca²⁺]_(i) levels.

In Vitro Kinase Assays

FLAG-tagged human kinases were immunoprecipitated from whole cell lysates of transiently-transfected HEK293 cells using anti-FLAG antibody-coupled protein G beads (Sigma), and immunoprecipitates were analysed for phosphorylation of either the entire NFAT1 regulatory domain (GST-NFAT1[1-415]) expressed in bacterial cells, or GST-fused peptides corresponding to the SRR-1 (amino acids 149-183), SP-2 (amino acids 206-237) and SP-3 (amino acids 264-295) motifs of NFAT1 (both wild-type and Ser→Ala mutants in serines phosphorylated in vivo)¹⁰. Immunocomplexes were washed twice with lysis buffer (1.0% NP-40, 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol [DTT], 20 mM β-glycerol-phosphate, 10 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 μg/ml aprotinin, 10 μg/ml leupeptin) and twice with kinase buffer (20 mM HEPES, pH 7.4, 20 mM MgCl₂, 1 mM DTT, 0.1 mM sodium orthovanadate, 20 mM β-glycerol-phosphate), and incubated at 30° C. for 20 minutes in a 40 μl final volume of kinase buffer in the presence of 20 μM ATP, 2 μCi [γ³²P]-ATP and 10 μg of wild-type or mutant GST-peptide substrate. Peptides were isolated on glutathione-sepharose and phosphorylation was assessed by SDS gel electrophoresis and autoradiography.

The ability of DYRK1A and DYRK2 to phosphorylate GST-NFAT1 fusion peptides was examined using 20 ng of recombinant protein kinase (Upstate Biotechnology) in a 40 μl final volume of kinase buffer in the presence of 20 μM ATP, 2 μCi [γ³²P]-ATP and 10 μg of GST-peptide substrate. The ability of GSK3 to phosphorylate NFAT1 was examined by first pre-phosphorylating GST fusion proteins pre-bound to glutathione sepharose beads using I U of recombinant protein kinase A (PKA) (New England Biolabs [NEB]), 20 ng DYRK1A or DYRK2 in the presence of 1 mM cold ATP for 16 h at 30° C. After cold priming fusion proteins were washed repeatedly to remove recombinant kinase and ATP. Phosphorylated fusion proteins were then incubated with 1 U of GSK3 (NEB) in a 40 μl final volume of kinase buffer in the presence of 20 μM ATP, 2 μCi [γ³²P]-ATP for 45 minutes.

Reporter Assays and IL-2 Expression Assays

Exponentially growing (10⁷) Jurkat T cells stably expressing HA-tagged full-length NFAT1 in the pOZ vector⁴² were transfected by electroporation at 250 V and 960 μF. For luciferase experiments, cells were transfected with 0.5 μg pRLTK reporter (Renilla luciferase for internal control), 5.0 μg pGL3 reporter (firefly luciferase, experimental promoter) and expression plasmids encoding empty vector, wild type or kinase dead DYRK2. At 24 h post transfection cells left untreated or stimulated with PMA (20 nM), ionomycin (1 μM) and 2 mM CaCl₂ for 6 hours were measured for reporter gene activity using the Dual-Luciferase Reporter Assay (Promega) as recommended by the manufacturer. For intracellular cytokine staining, cells were co-transfected with GFP-encoding plasmid and empty vector plasmids, wild type or kinase-dead DYRK2. At 24 h post transfection cells left untreated or stimulated with PMA (20 nM), ionomycin (1 μM) and 2 mM CaCl₂ for 6 hours in the presence of Brefeldin A (2 μg/mL) for the last 4 hours were fixed with 4% paraformaldehyde in PBS for 20 min at 25° C., washed twice with PBS, permeabilized in saponin buffer (PBS, 0.5% saponin [Sigma], 1% BSA and 0.1% sodium azide) and stained with phycoerythrin-conjugated rat anti-human IL-2 (PharMingen) for 30 min at 25° C. Cells were washed twice in PBS and analyzed with a FACSCalibur flow cytometer (Becton Dickinson) and FlowJo software.

siRNA-Mediated Knockdown of DYRK1A

0.5×10⁶ HeLa cells stably expressing NFAT1(1-460)-GFP were seeded in 6-well plates and transfected the next day with siRNAs (Dharmacon, Inc., Lafayette, Colo.) corresponding to control siRNA or human DYRK1A siRNA using lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's protocol. Cells were reseeded and the transfection procedure was repeated after 24 h to increase the efficiency of knockdown. Cells were harvested for immunoblot analysis or immunocytochemistry 4 days post transfection. DYRK transcript levels were measured by real-time RT-PCR. Threshold cycles (C_(T)) for DYRK1A were normalized to GAPDH housekeeping gene expression levels (ΔCT) and plotted as 0.5^(ΔCt)*10⁴ (arbitrary units). The siRNA sequences correspond to DYRK1A: AGGUGGAGGUGCAAUAUUA (SEQ ID NO: 31); scrambled control: CUUUAAGCCUCGAGAUAUA (SEQ ID NO: 32). The RT-PCR primer sequences correspond to DYRK1A sense: AGTTCTGGGTATTCCACCTGCTCA (SEQ ID NO: 10), DYRK1A anti-sense: TGAAGTTTACGGGTTCCTGGTGGT (SEQ ID NO: 11).

Intracellular Calcium Measurements by Time-Lapse Video Imaging

HEK 293T cells were grown directly on UV-sterilized coverslips, loaded with Ca²⁺ indicator dye Fura-2 AM (3 μM, Molecular Probes, Eugene, Oreg.) for 45 min at room temperature, washed and resuspended in loading medium (RPMI+10% FCS). For ratiometric Ca²⁺ videoimaging, coverslips were mounted on a closed bath RC-20 flow chamber (Warner Instrument Corp., Hamden, Conn.) and perfused in 2 mM Calcium Ringer solution (155 mM NaCl, 4.5 mM KCl, 10 mM D-glucose, 5 mM Hepes (pH 7.4), 1 mM MgCl₂, 2 mM CaCl₂). After switching to Ca²⁺ free Ringer solution (2 mM Ca²⁺ replaced with 2 mM MgCl₂), intracellular Ca²⁺ stores were depleted with 1 μM thapisgargin, and store-operated Ca²⁺ influx was measured after perfusing cells with Ringer solution containing 2 mM CaCl2. Single cell video imaging was performed on a S200 inverted epifluorescence microscope (Zeiss, Thornwood, N.Y.) using OpenLab imaging software (Improvision, Lexington, Mass.). Fura-2 emission was detected at 510 nm following excitation at 340 and 380 nm, respectively, with ratios of 340/380 being calculated for each 5 sec interval after background subtraction. Calibration values (R_(min), R_(max), S_(f)) were derived from cuvette measurements as previously described⁴³. For each experiment, approximately 50-100 cells were analyzed. For simultaneous measurements of [Ca²⁺]i and DYRK2 expression, Jurkat T cells were cotransfected with DYRK2 cDNA and eGFP at a ratio of 10:1. 48 hrs post transfection, cells were used for Ca²⁺ imaging as described above. For single cell analysis of [Ca²⁺]i, GFP⁻ (that is, DYRK2⁻) and GFP⁺ (that is, DYRK2⁺) cells were gated and plotted separately.

Intracellular Calcium Measurements by Flow Cytometry

S2R+ cells were detached from the dish with trypsin (CellGro, Hemdon, Va.) and loaded with the Ca²⁺ indicator dye Fluo4-AM (2 μM Molecular Probes, Eugene, Oreg.) for 45 min at room temperature and then resuspended in loading medium (RPMI+10% FCS). Immediately before the flow cytometric Ca²⁺ measurements, cells were resuspended in Ca²⁺ free Ringer solution and analyzed on a FACSCalibur (BD Biosciences, San Jose, Calif.). 180 sec after addition of thapsigargin (3 μM) in Ca²⁺ free Ringer to deplete intracellular Ca²⁺ stores, 4 mM Ca²⁺ Ringer solution was added to the cells to achieve a final concentration of 2 mM Ca²⁺. Cellular Ca²⁺ levels were then analyzed using FloJo software (Tree Star, Inc., Ashland, Oreg.).

Subcloning of Human Orthologues of the Candidate Kinases

Full-length cDNAs encoding human orthologues of the kinase candidates were obtained from Flexgene Kinase Repository (Harvard Institute of Proteomics)³⁶ or the Mammalian Gene Collection (MGC, Open Biosystems), subcloned into pENTRY.11 (Invitrogen) vectors with insertion of Flag-tag at the N-terminus, and then recombined into pDEST12.2 (Invitrogen). Kinase-dead DYRK2 was constructed by introducing a K251 R point mutation in the ATP binding pocket of the active site using the PCR-based method (QuikChange Site-Directed Mutagenesis, Stratagene) and sequenced to ensure polymerase fidelity.

Introduction and Results

The subcellular localization of NFAT is determined by a complex process of signal integration that involves inputs from diverse signalling pathways³⁻⁵. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca²⁺ ([Ca²⁺]₁) levels they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus 6. Dephosphorylation of NFAT by calcineurin is countered by distinct NFAT kinases, among them CK1, GSK3, and various members of the MAP kinase family^(3,7-10). The transcriptional activity of NFAT is regulated by additional inputs, including phosphorylation of the N-terminal transactivation domain, recruitment of co-activators and co-repressors, and choice of partner proteins in the nucleus^(3,9,11).

We used a strategy, based on genome-wide RNAi screening in Drosophila S2R+ cells 12-14, to identify regulators of intracellular free Ca²⁺ ([Ca²⁺]₁) levels, calcineurin activation and NFAT localization in cells. The strategy relies on the fact that although Ca²+-regulated NFAT proteins are not represented in Drosophila, the pathways of Ca²⁺ homeostasis, Ca²⁺ influx, and calcineurin activity that regulate NFAT localization are evolutionarily conserved^(15,16). To validate this point, we used the GFP fusion protein NFAT1(1-460)-GFP (here termed NFAT-GFP)¹⁷. NFAT-GFP contains the entire regulatory domain of NFAT, including the calcineurin and CK1 docking sites, the nuclear localization signal (NLS), and the conserved serine-rich regions (SRR) and serine-proline repeat (SP) motifs which control NFAT1 subcellular localization and DNA-binding affinity^(3,9,10,17) (FIG. 6A). NFAT-GFP was correctly regulated in Drosophila S2R+ cells: it was phosphorylated and properly localized to the cytoplasm under resting conditions and became dephosphorylated and translocated to the nucleus in response to Ca²⁺ store depletion with the SERCA inhibitor thapsigargin (FIG. 6B); it was imported into the nucleus with similar kinetics in S2R+ cells and mammalian HeLa cells and was sensitive to the calcineurin inhibitor CsA in both cell types. S2R+ cells treated with limiting amounts of thapsigargin displayed intermediate phosphorylated forms of NFAT-GFP, most likely reflecting progressive dephosphorylation of serines within the individual conserved motifs of the regulatory domain^(9,10). Finally, depletion of the primary NFAT regulator, calcineurin, by RNAi in S2R+ cells inhibited thapsigargin-dependent dephosphorylation and nuclear import of NFAT-GFP (Table II). Together these experiments confirmed that the major pathways regulating NFAT phosphorylation and subcellular localization—store-operated Ca²⁺ influx, calcineurin activation, and NFAT phosphorylation—are conserved in Drosophila and appropriately regulate vertebrate NFAT.

We performed a genome-wide RNAi screen^(12,13) on unstimulated S2R+ cells, and scored visually for aberrant nuclear localization of NFAT-GFP (see Methods and Example 3). Of 21,884 screened wells, 662 were scored as potentially positive using non-stringent criteria; in a confirmatory screen, 271/325 (83%) retested candidates were confirmed as positive, attesting to the reproducibility of our initial assessment of NFAT nuclear localization (FIG. 6C). Positive candidates included Na⁺/Ca²⁺ exchangers and SERCA Ca²⁺ ATP-ases whose knockdown would be expected to increase basal [Ca²⁺]₁, and the scaffold protein Horner which has been linked to Ca²⁺ influx and Ca²⁺ homeostasis^(18,19) (Table 1). The screen also identified Stim, a recently-identified regulator of store-operated Ca²⁺ influx²⁰⁻²² as causing nuclear localization of NFAT-GFP in resting S2R+ cells, possibly because its depletion resulted in minor dysregulation of NFAT kinases or small increases in basal [Ca²⁺]₁ levels (FIGS. 9A-9C). Finally, the screen identified a large number of protein kinases which could potentially influence basal [Ca²⁺], levels or calcineurin activity, directly phosphorylate the NFAT regulatory domain, or indirectly influence the activity of direct NFAT kinases (Table I).

We were interested in kinases that directly phosphorylate the NFAT regulatory domain. In the family member NFAT1, the regulatory domain bears>14 phosphorylated serines, 13 of which are dephosphorylated by calcineurin⁹ (FIG. 6A). Five of these serines are located in the SRR-1 motif, which controls exposure of the NLS and is a target for phosphorylation by CK1^(3,10); three are located in the SP-2 motif, which can be phosphorylated by GSK3 after a priming phosphorylation by protein kinase A (PKA)^(7,10); and four are located in the SP-3 motif, for which a relevant kinase had yet to be identified at the time this study was initiated. The SP-2 and SP-3 motifs do not directly regulate the subcellular localization of NFAT1, but their dephosphorylation increases both the probability of NLS exposure and the affinity of NFAT for DNA^(3,10,23). It was not known how distinct SRR-1, SP-2 and SP-3 kinases acted together to promote the full phosphorylation of NFAT; nevertheless, we expected that depletion of individual NFAT kinases in S2R+ cells would result in varying degrees of nuclear accumulation of NFAT, depending on kinase expression level, the particular motif phosphorylated, and whether or not other related kinases were redundantly expressed. We therefore tested at least one mammalian homologue (where available) of all constitutively-active kinases identified in the screen, regardless of their score in the secondary screen. Some inducible kinases were included, but others (e.g. protein kinases C and D) will be investigated as part of a separate study.

FLAG-tagged mammalian homologues of selected Drosophila kinases were expressed in HEK293 cells, and anti-FLAG immunoprecipitates were tested in an in vitro kinase assay for their ability to phosphorylate the GST-NFAT1 (1-415) fusion protein (FIG. 7A). Three novel candidates—PRKG1, DYRK2 and IRAK4—showed strong activity in this assay (FIG. 7A, lanes 8, 13 and 15; CK1 isoforms CK1α and CK1ε were included as positive controls in lanes 1 and 2). PRKG1 was expressed at equivalent or higher levels than DYRK2 (FIG. 7A, bottom panel, lanes 8 and 13), but only DYRK2 could counter the dephosphorylation of NFAT-GFP by calcineurin (FIG. 7B, lanes 3, 4; 7, 8; 11, 12). IRAK4 was poorly expressed (FIG. 7A, bottom panel, lane 15); however CD4+ Th1 cells isolated from IRAK4−/− mice showed normal NFAT1 dephosphorylation, rephosphorylation and nuclear transport compared to control cells. For these reasons, neither PRKG1 nor IRAK4 were further investigated.

We focused on the role of DYRK-family kinases as direct regulators of NFAT. Overexpression of DYRK2 maintained NFAT-GFP in its phosphorylated form after ionomycin treatment (FIG. 7B, lanes 5-8); similarly, overexpression of wild type (WT) DYRK2 but not a kinase-dead (KD) mutant of DYRK2, prevented NFAT nuclear localization in thapsigargin-treated cells. DYRK overexpression yielded a slower-migrating form of NFAT (FIG. 7B, lanes 7, 8), leading to the concern that DYRK (a serine/proline-directed kinase²⁴) phosphorylated SPRIEIT, the calcineurin docking sequence on NFAT1^(3,6), preventing NFAT:calcineurin interaction. However, DYRK2 inhibited the ionomycin-induced dephosphorylation of NFAT-GFP containing a SPRIEITPS>HPVIVITGP (VIVIT) substitution¹⁷, which eliminates the SP and TP sequences that could be targeted by DYRK. The ability of DYRK to inhibit dephosphorylation of the VIVIT-substituted NFAT-GFP is particularly impressive, given the higher affinity (˜40-50-fold) of the VIVIT docking site for calcineurin compared to the affinity of the wild type SPRIEIT (SEQ ID NO: 33) docking site¹⁷. Consistent with direct phosphorylation of NFAT, Ca²⁺ mobilization in response to thapsigargin was unaffected by depletion of the DYRK-family candidate CG40478 in S2R+ cells, and only slightly diminished by DYRK2 overexpression in Jurkat T cells.

DYRKs constitute an evolutionarily-conserved family of proline or arginine-directed protein kinases distantly related to cyclin-dependent kinases (CDK), mitogen-activated protein kinases (MAPK), glycogen synthetase kinases (GSK), and CDK-like (LK) kinases (CMGC kinases²⁴. The DYRK family has multiple members (FIG. 11A) which have been designated class I (nuclear, DYRK1A and DYRK1B) or class II (cytoplasmic, DYRK2-6), depending on their subcellular localisation²⁵⁻²⁶. RT-PCR and western blotting suggested that DYRK1A and DYRK2 were major representatives of nuclear and cytoplasmic DYRKs in Jurkat T cells, respectively (FIG. 11B). Depletion of endogenous DYRK1A using DYRK1A-specific siRNA in HeLa cells stably expressing NFAT-GFP increased the rate and extent of NFAT1 dephosphorylation and nuclear import while slowing rephosphorylation and nuclear export, in response to treatment with thapsigargin for 10 min (to induce dephosphorylation and nuclear import) followed by CsA addition for 5 to 30 min (to inactivate calcineurin and permit rephosphorylation by NFAT kinases for nuclear export) (FIG. 10C left panel). Results obtained using endogenous DYRK1A depletion, which reflect a knockdown efficiency of approximately 70% of mRNA levels (FIG. 10C right panel), indicate that DYRK represent physiological negative regulators of NFAT activation in cells.

Further experiments showed that DYRK specifically targeted the SP-3 motif of NFAT1. FLAG-tagged DYRK2 was expressed in HEK 293 cells, immunoprecipitated with anti-FLAG antibodies, and phosphorylated peptides corresponding to the conserved SP-3 but not the SP-2 motif of the NFAT regulatory domain in vitro. To rule out the possibility that the NFAT kinase was not DYRK itself but rather a DYRK-associated kinase, we tested bacterially-expressed recombinant DYRK1A and DYRK2 for in vitro phosphorylation of peptides corresponding to three conserved serine-rich motifs of NFAT1 phosphorylated in cells (SRR-1, SP-2 and SP-3 motifs⁹). DYRK2 and DYRK1A both displayed strong and selective kinase activity towards the SP-3 motif of NFAT1, but neither kinase phosphorylated an SP-3 peptide with Ser>Ala substitutions in the specific serine residues known to be phosphorylated in cells⁹. At least 2 serine residues (bold and underlined) in the SP-3 motif (SPQRSRSPSPQPSPHVAPQDD) (SEQ ID NO: 34) fit the known sequence preference of DYRK kinases for serine/threonine residues with arginine at the −2 or −3 position, and proline (or valine) at the +1 position²⁷⁻²⁹, and both are known to be phosphorylated in cells⁹ (see FIG. 6A). Additional studies will be needed to establish whether the two other phosphorylated serine residues (underlined) in the SP-3 motif are targets for DYRK or other NFAT kinases in vivo.

Phosphorylation at the SP-2 and SP-3 motifs are the primary determinants for upward mobility shift of phosphorylated NFAT1, and we have shown here and previously that they are phosphorylated by GSK3 and DYRK, respectively 9. Because DYRK kinases have been reported to prime for GSK3-mediated phosphorylation of protein-synthesis initiation factor eIF2Bε and the microtubule-associated protein tau²⁹, we asked whether DYRK kinases could similarly prime for GSK3-mediated phosphorylation of NFAT. The SP2 motif of NFAT1 can be phosphorylated by GSK3¹⁰, and GSK3 recognition of the target sequence requires a priming phosphorylation that can be mediated by PKA. In contrast to the strong priming by PKA, neither DYRK2 nor DYRK1A could efficiently prime for phosphorylation of the SP-2 motif by GSK3.

As DYRK2 phosphorylated only the SP-3 motif of NFAT in vitro, and because it was not a priming kinase for GSK3 at the SP-2 motif, we expected that it would cause only half the expected mobility shift of NFAT1 when expressed in cells. However, overexpression of DYRK2 resulted in complete phosphorylation of NFAT1 (FIG. 7B). To resolve this paradox, we asked whether prior phosphorylation of the entire NFAT regulatory domain by DYRK would facilitate further phosphorylation by GSK3. The GST-NFAT1(1-415) fusion protein was prephosphorylated to completion by PKA or DYRK2 using the recombinant kinases, then washed and incubated briefly (45 min) in the absence or presence of recombinant GSK3 and radiolabelled [γ-³²P] ATP. As shown previously, GSK3 does not phosphorylate GST-NFAT1 (1-415) without priming, but does phosphorylate after pre-phosphorylation with either PKA or DYRK2. Pre-phosphorylation with DYRK2 caused an upward mobility shift of the GST-NFAT1 (1-415) substrate as judged by Coomassie blue staining, as expected from the fact that DYRK2 phosphorylates the SP-3 motif; moreover, pre-phosphorylation with DYRK2 yielded a radioactive GSK3-phosphorylated band of slower mobility compared to the band observed after pre-phosphorylation with PKA. These results suggest that while PKA primes for GSK3 by phosphorylating the fourth serine (bold) in the SP-2 motif (SPRTSPIMSPRTSLAED) (SEQ ID NO: 35) and permitting processive N-terminal phosphorylation of the underlined serines by GSK3, while DYRK2 potentiates GSK3-mediated phosphorylation of the regulatory domain motif by phosphorylating a separate motif, the SP-3 motif. Indeed, the serine targeted by PKA in the SP-2 motif is not found phosphorylated in cells¹⁰, providing further evidence for physiological regulation of NFAT by DYRK.

We asked whether DYRK expression regulated the transcriptional activity of NFAT utilizing the kinase-dead mutant of DYRK2 as an inhibitor of DYRK activity in cells^(30,31). Jurkat T cells were co-transfected with an IL-2 promoter-driven luciferase reporter plasmid and increasing amounts of expression plasmids for either wild type (WT) or kinase-dead (KD) DYRK2; one day later, the cells were stimulated for 6 h with PMA and ionomycin and reporter activity was measured. WT DYRK2 strongly diminished NFAT-dependent activity, while the KD mutant behaved as an inhibitor by increasing NFAT-dependent luciferase activity at higher concentrations (FIG. 8A). Similar results were obtained using luciferase reporters containing tandem copies of the ARRE2 NFAT:AP-1 site of the IL-2 promoter³² as well as the κ3 site of the TNFα promoter³³. In related experiments expression WT DYRK2 also diminished, the production of endogenous IL-2 by stimulated Jurkat T cells in a dose-dependent manner while KD DYRK2 again had an inhibitory effect, when expressed at high concentrations, by increasing IL-2 production under these conditions (FIGS. 8B, 8C). Furthermore, we detected endogenous DYRK2 co-immunoprecipitating with HA-NFAT1 stably expressed at low endogenous levels in a Jurkat cell line; in this respect DYRK may resemble the SRR-1 kinase CK1, which forms a stable complex with NFAT under resting conditions but dissociates following activation¹⁰. A DYRK-NFAT interaction supports the hypothesis that DYRK is a physiological NFAT kinase: kinase-substrate interactions of this type are known to be critical in many other signal transduction pathways, although they are often transient and difficult to detect at endogenous levels of expression³⁴.

Discussion

We have shown that genome-wide RNAi screening in Drosophila is a valid and powerful strategy for exploring novel aspects of signal transduction in mammalian cells, provided that key members of the signaling pathway are evolutionarily conserved and represented in the Drosophila genome. We have used the method to identify conserved regulators of the purely vertebrate transcription factor, NFAT; to our knowledge, this is the first example of a genome-wide RNAi screen that crosses evolutionary boundaries in this manner. The strategy was successful because Drosophila developed an evolutionary niche that was later used by Ca²⁺-regulated NFAT proteins when they emerged in vertebrates. Using this approach we have identified DYRK as a novel physiological regulator of NFAT, and the first SP-3 motif-directed kinase. It is likely that conserved aspects of the regulation of other mammalian processes will also be successfully defined by developing assays in Drosophila cells.

Our data suggest that DYRK regulates NFAT phosphorylation by a mechanism in which DYRK phosphorylates the NFAT regulatory domain within the conserved SP-3 motif, and thereby facilitates further phosphorylation of the NFAT regulatory domain by GSK3. A similar sequential mechanism may regulate progressive dephosphorylation of NFAT, whereby dephosphorylation of the SRR-1 motif promotes dephosphorylation of the SP-2 and SP-3 motifs by increasing their accessibility to calcineurin⁹. It is likely that class II DYRKs (DYRK2, 3 and 4) which are localized to the cytoplasm²⁵, function primarily as “maintenance” kinases that sustain the phosphorylation status of cytoplasmic NFAT in resting cells, whereas class I DYRKs (DYRK1A and 1B), which are localized to the nucleus²⁵, re-phosphorylate nuclear NFAT and promote its nuclear export. Notably, DYRK1A and the endogenous calcineurin regulator RCN/DSCR1/calcipressin-1 are both localized to the Down Syndrome Critical Region on chromosome 21. Thus overexpression of these negative regulators of NFAT in Down Syndrome could contribute, by inhibiting NFAT activation, to the severe neurological and immune developmental defects associated with chromosome 21 trisomy³⁵.

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Example 3 Table I

List of candidates that were positive in the secondary screen, classified into the categories in Table I. The first column indicates whether or not the candidate was retested in the confirmatory screen (NT, not tested); if tested, the summed localization score from 3 separate experiments is shown (see Methods). Other columns list gene names, Flybase numbers, and human orthologues as obtained from Homologene (for the kinase category, the phylogenetic analysis described in Methods was used in addition), and number of predicted off-targets with exact match of 21-nt, 37 candidates with >10 off-targets are not listed.

Table II.

Analysis of expression, RNAi phenotype in thapsigargin-treated cells, and amplicon off-targets for calcineurin subunits and related proteins. Expression level of the subunits in S2R+ cells was estimated by RT-PCR analysis, and the effect of their depletion on NFAT nuclear localization in thapsigargin (TG)-treated cells was evaluated (+++, strong inhibition; -, no inhibition). The DRSC amplicons targeting each of the subunits were analyzed for predicted off-targets with exact matches of 21-, 20-, or 19-nt as described in Methods. Description of the off-targets is provided in Table III. Red indicates off-targets belonging to the same family as the primary targets.

Of the three isoforms of calcineurin A, the amplicon for CanA1 and one amplicon each for Pp2b-1 4D and CanA-14F show no predicted off-targets. CanA1 is poorly expressed and its depletion does not inhibit NFAT nuclear translocation, while Pp2B-1 4D and CanA-14F are both expressed and depletion of either isoform results in strong inhibition of NFAT nuclear translocation.

Why does depletion of the moderately expressed isoform CanA-14F give similar inhibition as depletion of the more highly expressed isoform Pp2B-14D? Different methods have different sensitivities, and while the eye is able to discern subtle changes in the nuclear localization of NFAT, such visual estimates are not as quantitative as (for instance) estimating extent of dephosphorylation by western blotting.

Of the three isoforms of calcineurin B, two (CanB and CanB2) are strongly related to mammalian calcineurin B while CG32812 is more distantly related, resembling mammalian CHP. RNAi against either CanB or CanB2 gave equivalent inhibition (˜700%) of NFAT nuclear localization, even though CanB is barely expressed while CanB2 is expressed at high levels. This is most likely due to the fact that CanB and CanB2 are reciprocal off-targets, with 20 nt overlap in their respective amplicons DRSC 18449 and DRSC07355.

Table IV.

Amplicon off-targets for selected candidates that were evaluated in additional experiments. Scores of the candidates in the confirmatory screen, evaluating the effects of their RNAi-mediated depletion on NFAT nuclear accumulation in resting cells, are shown (taken from Table 1). For each candidate with positive DRSC amplicons, predicted off-targets with exact matches of 21-, 20-, or 19-nt are listed. Description of the off-targets is provided below. Red indicates off-targets belonging to the same family as the primary targets that were positive in the initial screen.

The amplicon corresponding to the GSK3 homologue sgg (DRSC18832) gave the highest score but also has a high number of off-targets. None of these off-targets corresponds to gskt (DRSC14056), which gave a low score of 1 in the primary screen.

The amplicon corresponding to the highest-scoring CK1 family membergish has no predicted off-targets, indicating that it represents a bonafide regulator of NFAT. Clear cross-inactivation exists for amplicons DRSC16929, DRSC20231 and DRSC19863, corresponding to the CK1 isoforms dco, CK1alpha/CG2028 and CG2577, each of which has a positive localization score of 1. Further work is necessary to determine whether the scores associated with the other isoforms reflect expression levels of the isoforms, off-target effects, or both.

We are fortunate that for the two candidates—DYRK and STIM—that we focused on for this study, there are no predicted off-targets for exact matches of either 21, 20 or 19 nt.

TABLE 1 Number of Human Score in secondary potential 21 nt orthologs (NCBI Description of the human screen Gene FBGN off-targets Homologene) orthologs (NCBI Gene) PHOSPHATASES 5 Ptip FBgn0039111 0 PTPMT1 protein tyrosine phosphatase, mitochondrial 1 3 CanA1 FBgn0010015 0 PPP3CC protein phosphatase 3 (formerly 2B), catalytic subunit, gamma isoform 3 flw FBgn0000711 1 PPP1CB protein phosphatase 1, catalytic subunit, beta isoform 3 PpD6 FBgn0005779 1 3 wob FBgn0027492 0 PPP2R5E protein phosphatase 2, regulatory subunit B (B56), epsilon isoform 1 CanB FBgn0010014 0 PPP3R1 protein phosphatase 3 (formerly 2B), regulatory subunit B, 19 kDa, alpha isoform 1 CanB2 FBgn0015614 0 PPP3R1 protein phosphatase 3 (formerly 2B), regulatory subunit B, 19 kDa, alpha isoform 1 CG32812 FBgn0025642 0 LOC63928 hepatocellular carcinoma antigen gene 520/ related to mammalian CHP 0 Pp2B-14D FBgn0011826 1 PPP3CB protein phosphatase 3 (formerly 2B), catalytic subunit, beta isoform PROTEIN KINASES 6 sgg FBgn0003371 3 GSK3B glycogen synthase kinase 3 beta 5 CG7125 FBgn0038603 0 PRKD protein kinase D 4 CG31640 FBgn0051640 0 DDR 4 gish FBgn0011253 0 CSNK1G casein kinase 1, gamma 4 inaC FBgn0004784 0 PRKCB1 protein kinase C, beta 1 3 CG12147 FBgn0037325 0 CSNK1 casein kinase 1 family 3 CkIIalpha FBgn0000258 0 CSNK2A1.2 casein kinase 2, alpha 3 pII FBgn0010441 0 IRAK 2 CG2905, FBgn0004661 0 TRRAP transformation/transcription domain- Nipped-A associated protein 2 aPKC FBgn0022131 0 PRKCI protein kinase C, iota 2 CG11489 FBgn0025702 0 SRPK1 SFRS protein kinase 1 2 CG32687 FBgn0052687 0 LOC116064 hypothetical protein LOC116064 2 CG6498 FBgn0036511 0 MAST2 microtubule associated serine/threonine kinase 2 2 CG7097 FBgn0034421 0 MAP4K3 mitogen-activated protein kinase kinase kinase kinase 3 2 I(1)G0148 FBgn0028360 0 CDC7 CDC7 cell division cycle 7 2 Pkc53E FBgn0003091 0 PRKCA protein kinase C, alpha 2 Pkcdelta FBgn0030387 0 PRKCD protein kinase C, delta 2 polo FBgn0003124 0 PLK1 polo-like kinase 1 2 trc FBgn0003744 0 STK38, STK38L serine/threonine kinase 38 like 1 CG40478 FBgn0069975 0 DYRK dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1 CG2577 FBgn0030384 3 CSNK1 casein kinase 1 family 1 CG4168 FBgn0028888 0 1 CG5483 FBgn0038816 0 1 CG7094 FBgn0032650 0 CSNK1 casein kinase 1 family 1 CkIalpha FBgn0015024 3 CSNK1A1 casein kinase 1, alpha 1 1 Cks FBgn0010314 0 CKS1B CDC28 protein kinase regulatory subunit 1B 1 dco FBgn0002413 0 CSNK1D, E casein kinase 1, delta/epsilon 1 for FBgn0000721 2 PRKG1 protein kinase, cGMP-dependent, type I 1 gskt FBgn0046332 0 GSK3A 1 phl FBgn0003079 2 BRAF v-raf murine sarcoma viral oncogene homolog B1 1 Pk61C FBgn0020386 0 PDPK1 3-phosphoinositide dependent protein kinase 1 1 Pkc98E FBgn0003093 0 PRKCE protein kinase C, epsilon 1 Tie FBgn0014073 4 0 CG11533 FBgn0039908 0 0 CG9962 FBgn0031441 0 CSNK1 casein kinase 1 family 0 CG10579 FBgn0005640 0 ALS2CR7, PFTK1 PFTAIRE protein kinase 1 0 Png FBgn0000826 0 NT CG17698 FBgn0040056 0 CAMKK2 calcium/calmodulin-dependent protein kinase kinase 2, beta NT gek FBgn0023081 0 CDC428PA, B CDC42 binding protein kinase alpha (DMPK- like) OTHER KINASES/ KINASE-RELATED 1 Pi3K59F FBgn0015277 0 PIK3C3 phosphoinositide-3-kinase, class 3 0 CG8298 FBgn0033673 0 0 Pdk FBgn0017558 0 PDK3 pyruvate dehydrogenase kinase, isoenzyme 3 NT CG3809 FBgn0037995 0 NT CG6218 FBgn0038321 0 NAGK N-acetylglucosamine kinase NT CG6364 FBgn0039179 0 UCK2 uridine-cytidine kinase 2 NT dlg FBgn0001624 8 DLG1 discs, large homolog 1 MISCELLANEOUS/ CALCIUM- RELATED 5 CG14387 FBgn0038089 0 4 TpnC4 FBgn0033027 0 4 TpnC73F FBgn0010424 0 3 Stim FBgn0045073 0 STIM1 stromal interaction molecule 1 3 Cam FBgn0000253 0 CALM2 calmodulin 2 (phosphorylase kinase, delta) 3 CG11165 FBgn0033238 2 3 CG13898 FBgn0035161 0 2 norPA FBgn0004625 0 PLCB4 phospholipase C, beta 4 2 TpnC41C FBgn0013348 0 2 TpnC47D FBgn0010423 0 1 CG13526 FBgn0034774 0 1 CG31345 FBgn0051345 0 CAPSL calcyphosine-like 1 CG31650 FBgn0031673 0 RCN2 reticulocalbin 2, EF-hand calcium binding domain 1 CG31958 FBgn0051958 2 1 CG31960 FBgn0051960 2 1 TpnC25D FBgn0031692 1 MEMBRANE SIGNALLING 5 CG6919 FBgn0038980 0 HTR4 5-hydroxytryptamine (serotonin) receptor 4 4 CG30340 FBgn0050340 0 4 DopR FBgn0011582 4 DRD1 dopamine receptor D1 4 Gr47a FBgn0041242 0 4 Or85d FBgn0037594 0 4 Su(fu) FBgn0005355 0 SUFU suppressor of fused homolog (Drosophila) 3 Ac3 FBgn0023416 0 ADCY3 adenylate cyclase 3 3 Gyc-89Db FBgn0038436 0 3 homer FBgn0025777 0 HOMER2 homer homolog 2 3 mav FBgn0039914 0 TGFB3 transforming growth factor, beta 3 3 PGRP-LE FBgn0030695 0 PGLYRP3 peptidoglycan recognition protein 3 2 cenB1A FBgn0039056 0 CENTB2 centaurin, beta 2 2 CG10823 FBgn0038880 0 2 CG11319 FBgn0031835 0 DPP10 dipeptidylpeptidase 10 2 CG6989 FBgn0038063 0 2 fz3 FBgn0027343 0 2 N FBgn0004647 0 NOTCH1 Notch homolog 1, translocation-associated 2 Plc21C FBgn0004611 0 PLCB1 phospholipase C, beta 1 (phosphoinositide- specific) 2 pxb FBgn0053207 1 2 sog FBgn0003463 0 CHRD chordin 2 spz FBgn0003495 0 1 18w FBgn0004364 0 1 CG16752 FBgn0029768 0 1 CG17262 FBgn0031499 0 1 Crag FBgn0025864 0 MYCPBP c-myc promoter binding protein 1 Grip FBgn0040917 0 GRIP1 glutamate receptor interacting protein 1 1 nkd FBgn0002945 0 1 sl FBgn0003416 0 PLCG1 phospholipase C, gamma 1 0 bm FBgn0000221 0 B3GALT2 UDP-Gal:betaGlcNAc beta 1,3- galactosyltransferase, polypeptide 2 0 CG10747 FBgn0032845 0 PLCXD2 phosphatidylinositol-specific phospholipase C, X domain containing 2 0 CG31350 FBgn0051350 2 0 fz2 FBgn0016797 0 FZD8 frizzled homolog 8 0 Rab-RP1 FBgn0015788 0 RAB32 RAB32, member RAS oncogene family 0 skf FBgn0050021 0 MPP7 membrane protein, palmitoylated 7 NT Alg10 FBgn0052076 0 NT CG30361 FBgn0050361 4 GRM4 glutamate receptor, metabotropic 4 NT rho-5 FBgn0041723 0 NT Sema-1a FBgn0011259 0 SEMA6D sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D NT sif FBgn0019652 0 NT Syx1A FBgn0013343 0 STX1A syntaxin 1A NT tinc FBgn0038554 0 CATION CHANNELS AND TRANSPORTERS 5 CG13223 FBgn0033599 0 SLC24A6 solute carrier family 24 (sodium/potassium/calcium exchanger), member 6 5 CG14741 FBgn0037989 0 ATP8B2 ATPase, Class I, type 8B, member 2 4 CG10465 FBgn0033017 0 KCTD10 potassium channel tetramerisation domain containing 10 4 CG6737 FBgn0032294 0 4 Cng FBgn0014462 0 CNGA3 cyclic nucleotide gated channel alpha 3 4 GluRIIA FBgn0004620 0 4 inx6 FBgn0027107 0 4 Irk3 FBgn0032706 0 3 Ca-beta FBgn0015608 4 3 Ca-P60A FBgn0004551 0 ATP2A1 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 3 CG11155 FBgn0039927 0 GRIK3 glutamate receptor, ionotropic, kainate 3 3 CG2165 FBgn0025704 0 ATP2B3 ATpase, Ca++ transporting, plasma membrane 3 3 CG32792 FBgn0052792 0 3 CG3367 FBgn0029871 2 3 CG4450 FBgn0032113 0 3 CG6812 FBgn0036843 0 SFXN2 sideroflexin 2 3 KaiRIA FBgn0028422 1 GRIA4 glutamate receptor, ionotrophic, AMPA 4 3 ppk21 FBgn0039675 0 3 trp FBgn0003861 0 2 Ca-alpha1D FBgn0001991 0 CACNA1D calcium channel, voltage-dependent, L type, alpha 1D subunit 2 Catx FBgn0013995 0 SLC8A3 solute carrier family 8 (sodium-calcium exchanger), member 3 2 CG12376 FBgn0033323 0 SLC24A6 solute carrier family 24 (sodium/potassium/calcium exchanger), member 6 2 CG12904 FBgn0033510 0 KCNT2 potassium channel, subfamily T, member 2 2 CG1698 FBgn0033443 1 2 CG31284 FBgn0051284 0 2 CG31729 FBgn0051729 0 ATP9B ATPase, Class II, type 9B 2 CG3822 FBgn0038837 0 GRIK1 glutamate receptor, ionotropic, kainate 1 2 CG4536 FBgn0029904 5 2 CG9361 FBgn0037690 0 KCNK9 potassium channel, subfamily K, member 9 2 elk FBgn0011589 0 KCNH8 potassium voltage-gated channel, subfamily H (eag-related), member 8 2 GluClalpha FBgn0024963 0 GLRA3 glycine receptor, alpha 3 2 GluRIII FBgn0031293 0 2 Irk2 FBgn0039081 0 KCNJ9 potassium inwardly-rectifying channel, subfamily J, member 9 2 KCNQ FBgn0033494 3 KCNQ5 potassium voltage-gated channel, KQT-like subfamily, member 5 2 nAcRalpha-34E FBgn0028875 0 CHRNA7 cholinergic receptor, nicotinic, alpha polypeptide 7 2 nAcRalpha- FBgn0000036 0 CHRNA3 cholinergic receptor, nicotinic, alpha 96Aa polypeptide 3 2 Nmdar1 FBgn0010399 1 GRIN1 glutamate receptor, ionotropic, N-methyl D- aspartate 1 2 Ork1 FBgn0017561 0 KCNK4 potassium channel, subfamily K, member 4 2 sei FBgn0003353 0 KCNH6 potassium voltage-gated channel, subfamily H (eag-related), member 6 1 Ca-alpha1T FBgn0029846 0 1 cac FBgn0005563 0 CACNA1A calcium channel, voltage-dependent, P/Q type, alpha 1A subunit 1 CG10830 FBgn0038839 0 KCTD12 potassium channel tetramerisation domain containing 12 1 CG31201 FBgn0051201 1 GRIA4 glutamate receptor, ionotrophic, AMPA 4 1 CG32770 FBgn0052770 0 1 CG33298 FBgn0032120 0 ATP10A ATPase, Class V, type 10A 1 CG40146 FBgn0039941 0 1 CG5621 FBgn0038840 0 1 CG8743 FBgn0036904 0 MCOLN3 mucolipin 3 1 CG9935 FBgn0039916 1 GRIA1 glutamate receptor, ionotropic, AMPA 1 1 eag FBgn0000535 0 KCNH1 potassium voltage-gated channel, subfamily H 1 Glu-RIB FBgn0028431 1 GRIA2 glutamate receptor, ionotropic, AMPA 2 1 GluRIIB FBgn0020429 0 1 Ir FBgn0039061 0 KCNJ5 potassium inwardly-nectifying channel, subfamily J, member 5 1 I(2)01810 FBgn0010497 0 1 nAcRalpha- FBgn0000039 1 CHRNA2 cholinergic receptor, nicotinic, alpha 96Ab polypeptide 2 1 nAcRbeta-64B FBgn0000038 0 CHRNA4 cholinergic receptor, nicotinic, alpha polypeptide 4 1 nAcRbeta-96A FBgn000411B 0 CHRNB4 cholinergic receptor, nicotinic, beta polypeptide 1 Nmdar2 FBgn0014432 0 GRIN2B glutamate receptor, ionotropic, N-methyl D- aspartate 2B 1 nompC FBgn0016920 0 1 pain FBgn0060296 0 1 Pkd2 FBgn0041195 0 PKD2L1 polycystic kidney disease 2-like 1 1 Shal FBgn0005564 0 KCND3 potassium voltage-gated channel, Shal- related subfamily, member 3 1 Sip1 FBgn0010620 0 TFIP11 tuflelin interacting protein 11 1 sio FBgn0003429 0 KCNMA1 potassium large conductance calcium- activated channel, subfamily M, alpha member 1 0 Anktm1/TrpA1 FBgn0035934 0 TRPA1 transient receptor potential cation channel, subfamily A, member 1 0 CG12455 FBgn0028859 0 CACNA2D3 calcium channel, voltage-dependent, alpha 2/delta 3 subunit 0 CG13762 FBgn0040333 1 PKD2L1 polycystic kidney disease 2-like 1 0 CG14647 FBgn0037244 0 KCTD9 potassium channel tetramerisation domain containing 9 0 CG17922 FBgn0034656 0 CNGB1 cyclic nucleotide gated channel beta 1 0 CG32704 FBgn0052704 0 0 CG32810 FBgn0025394 0 KCTD5 potassium channel tetramerisation domain containing 5 0 CG4301 FBgn0030747 0 ATP11B ATPase, Class VI, type 11B 0 CG9472 FBgn0036874 0 PKD1L3 polycystic kidney disease 1-like 3 0 clumsy FBgn0026255 0 GRIK2 glutamate receptor, ionotropic, kainste 2 0 cngl FBgn0029090 3 0 Glu-RI FBgn0004619 0 GRIA3 glutamate receptor, ionotrophic, AMPA 3 0 Nckx30C FBgn0028704 0 SLC24A2 solute carrier family 24 (sodium/potassium/calcium exchanger), member 2 0 Rya-r44F FBgn0011286 0 RYR2 ryanodine receptor 2 (cardiac) 0 Shab FBgn0003383 0 KCNB1 potassium voltage-gated channel, Shab- related subfamily, member 1 0 SK FBgn0029761 0 KCNN3 potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3 0 trpl FBgn0005614 0 NT CG2196 FBgn0039872 1 NT nAcRalpha-808 FBgn0037212 0 OTHER TRANSPORTERS 3 ATPsyn-CI6 FBgn0016119 0 3 CG1599 FBgn0033452 0 SYBL1 synaptobrevin-like 1 3 CG31116 FBgn0051116 0 CLCN2 chloride channel 2 3 CG31158 FBgn0051158 0 3 CG31305 FBgn0051305 0 SLC25A1 solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1 3 CG6901 FBgn0038414 0 3 Mst84Db FBgn0004173 0 2 CG3860 FBgn0034951 0 OSBPL1A oxysterol binding protein-like 1A 2 CG3902 FBgn0036824 1 ACADSB acyl-Coenzyme A dehydrogenase, short/branched chain 2 CG5127 FBgn0039335 0 2 CG7442 FBgn0037140 0 2 CG7578 FBgn0028538 0 ARFGEF1 ADP-ribosylation factor guanine nucleotide- exchange factor 1 2 CG9270 FBgn0032908 0 ABCC2 ATP-binding cassette, sub-family C (CFTR/MRP), member 2 1 CG31731 FBgn0028539 0 1 CG8389 FBgn0034063 0 1 rdgB FBgn0003218 0 PITPNM2 phosphatidylinositol transfer protein, membrane-associated 2 1 w FBgn0003996 0 0 CG33214 FBgn0053214 0 GLG1 golgi apparatus protein 1 0 CG7458 FBgn0037144 0 NT Beach1 FBgn0043362 0 WDFY3 WD repeat and FYVE domain containing 3 NT CG12539 FBgn0030586 0 NT CG14482 FBgn0034245 0 NT CG14691 FBgn0037829 0 SV2A synaptic vesicle glycoprotein 2A NT CG17119 FBgn0039045 0 CTNS cystinosis, nephropethic NT CG18324 FBgn0033905 0 SLC25A34 solute carrier family 25, member 34 NT CG3071 FBgn0023527 0 UTP15 UTP15, U3 small nucleolar ribonucleoprotein NT CG32230 FBgn0052230 0 NT CG6142 FBgn0039415 0 NT CG7181 FBgn0037097 0 NT CG7830 FBgn0032015 0 TUSC3 tumor suppressor candidate 3 NT CG9990 FBgn0039594 0 NT Cyp49a1 FBgn0033524 0 CYP27A1 cytochrome P450, family 27, subfamily A, polypeptide 1 NT didum FBgn0015933 0 MYO5A myosin VA (heavy polypeptide 12, myoxin) NT ERp60 FBgn0033663 1 PDIA3 protein disulfide isomerase-associated 3 NT Pbprp2 FBgn0011280 0 NT Syx6 FBgn0037084 3 STX10 syntaxin 10 MISCELLANEOUS/ OTHER 6 Prosalpha7 FBgn0023175 0 PSMA3 proteasome (prosome, macropain) subunit, alpha type, 3 5 CG3812 FBgn0030421 0 AGPAT1 1-acylglycerol-3-phosphate O- acyltransferase 1 (lysophosphatidic acid acyltransferase, alpha) 4 bif FBgn0014133 3 4 CG11727 FBgn0030299 0 4 CG2781 FBgn0037534 0 ELOVL7 ELOVL family member 7, elongation of long chain fatty acids 4 CG4960 FBgn0039371 0 C19orf32 chromosome 19 open reading frame 32 4 CG7304 FBgn0036527 0 GALNT11 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferase 11 (GalNAc T11) 4 CG8258 FBgn0033342 0 CCT8 chaperonin containing TCP1, subunit 8 (theta) 4 CRMP FBgn0023023 0 DPYS dihydropyrimidinase 4 Eip63F-1 FBgn0004910 0 3 Act57B FBgn0000044 5 ACTB actin, beta 3 CG11299 FBgn0034897 0 SESN3 sestrin 3 3 CG6509 FBgn0032363 0 DLG5 discs, large homolog 5 3 CG9342 FBgn0032904 0 MTP microsomal triglyceride transfer protein (large polypeptide, 88 kDa) 3 CG9467 FBgn0037758 0 KCTD3 potassium channel tetramerisation domain containing 3 3 eIF-2beta FBgn0004926 0 EIF2S2 eukaryotic translation initiation factor 2, subunit 2 beta, 38 kDa 3 fzo FBgn0011596 0 MFN1 milofusin 1 3 pros FBgn0004596 0 PROX1 prospero-related homeobox 1 3 Su(var)3-9 FBgn0003600 0 EIF2S3 eukaryotic translation initiation factor 2, subunit 3 gamma, 52 kDa 2 14-3-3epsilon FBgn0020238 0 YWHAE tyrosine 3-monoxygenase/tryptophan 5- monooxygenase activation protein, epsilon polypeptid 2 ac FBgn0000022 3 ASCL2 achaele-scute complex-like 2 2 Arp66B FBgn0011744 0 ACTR3 ARP3 actin-related protein 3 homolog 2 CG10069 FBgn0034611 0 SLC37A2 solute carrier family 37 (glycerol-3- phosphate transporter), member 2 2 CG11600 FBgn0038068 1 2 CG11608 FBgn0038069 0 LIPL3 lipase-like, ab-hydrolase domain containing 3 2 CG14625 FBgn0040358 4 2 CG2678 FBgn0014931 0 2 CG3074 FBgn0034709 0 TINAGL1 tubulointerstitial nephritis antigen-like 1 2 CG32635 FBgn0052635 1 2 CG4448 FBgn0039067 0 2 CG5278 FBgn0038986 3 2 CG5802 FBgn0038863 0 SLC35B1 solute carrier family 35, member B1 2 CG7140 FBgn0037147 0 2 Rad51D FBgn0030931 0 XRCC2 X-ray repair complementing defective repair in Chinese hamster cells 2 1 cer FBgn0034443 0 1 CG6330 FBgn0039464 0 UPP2 uridine phosphorylase 2 1 CG7568 FBgn0039673 0 WDR69 WD repeat domain 69. 1 CG9326 FBgn0032885 0 MPP6 membrane protein, pelmitoylated 6 (MAGUK p55 subfamily member 6) 1 CG9784 FBgn0030761 0 PIB5PA phosphatidylinositol (4,5) bisphosphate 5- phosphatase, A 1 cnc FBgn0000338 0 1 eIF2B-beta FBgn0024996 0 EIF2B2 eukaryotic translation initiation factor 2B, subunit 2 beta, 39 kDa 1 gammaTub23C FBgn0004176 0 TUBG1 tubulin, gamma 1 1 Hn FBgn0001208 0 PAH phenylalanine hydroxylase 1 Pgant35A FBgn0001970 0 GALNT11 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N- acetylgalactosaminyltransferase 11 (GalNAc T11) 1 pgant4 FBgn0051956 0 1 skpA FBgn0025637 1 LOC401713 organ of Corti protein 2; RNA polymerase II elongation factor-like protein OCP2; cyclin A/CDK2-associated p19 0 CG15408 FBgn0031523 0 0 CG4500 FBgn0028519 0 ACSBG1 acyl-CoA synthetase bubblegum family member 1 0 CG7348 FBgn0036940 0 0 CG9647 FBgn0035729 0 0 D FBgn0000411 1 0 nahode FBgn0034797 0 0 Pde6 FBgn0038237 0 PDE11A phosphodiesterase 11A 0 sdt FBgn0003349 1 MPP5 membrane protein, palmitoylated 5 (MAGUK p55 subfamily member 5) 0 TSG101 FBgn0036666 0 TSG101 tumor susceptibility gene 101 NT Aats-cys FBgn0027091 0 CARS cysteinyl-tRNA synthetase NT Aats-met FBgn0027083 0 MARS2 methionine-tRNA synthetase 2 NT Acp70A FBgn0003034 0 NT Act79B FBgn0000045 5 ACTG2 actin, gamma 2, smooth muscle, enteric NT Ahcy13 FBgn0014455 0 AHCY S-adenosylhomocysteine hydrolase NT amon FBgn0023179 0 PCSK2 proprotein convertase subtilisin/kexin type 2 NT asparagine- FBgn0041607 0 synthetase NT ATbp FBgn0039946 5 NT BEAF-32 FBgn0015602 0 NT beat-Ic FBgn0028644 8 NT beat-Vb FBgn0038092 0 NT Bin1 FBgn0024491 0 SAP18 sin3-associated polypeptide, 18 kDa NT BM-40-SPARC FBgn0026562 0 SPARCL1 SPARC-like 1 (mast9, hevin) NT btsz FBgn0010940 0 NT bwa FBgn0045064 0 ASAH3L N-acylsphingosine amidohydrolase 3-like NT CG10168 FBgn0039087 0 NT CG11107 FBgn0033160 0 DHX15 DEAH (Asp-Glu-Ala-His) box polypeptide 15 NT CG12162 FBgn0037329 0 POLDIP2 polymerase (DNA-directed), delta interacting protein 2 NT CG13643 FBgn0040601 0 NT CG13779 FBgn0040954 0 NT CG14869 FBgn0038341 0 NT CG15105 FBgn0034412 0 NT CG1571 FBgn0029993 0 DNAI2 dynein, axonemal, intermediate polypeptide 2 NT CG16710 FBgn0039101 0 NT CG16857 FBgn0028482 0 NT CG17294 FBgn0032032 0 HDHD2 haloacid dehalogenase-like hydrolase domain containing 2 NT CG17826 FBgn0036227 0 FBN2 fibrillin 2 (congenital contractural arachnodactyly) NT CG18493 FBgn0038701 0 NT CG2051 FBgn0037376 0 HAT1 histone acetyltransferase 1 NT CG3066 FBgn0037515 0 NT CG31115 FBgn0051115 0 MTAP methylthioedenosine phosphorylase NT CG31159 FBgn0051159 0 GFM2 G elongation factor, mitochondrial 2 NT CG31224 FBgn0051224 0 NT CG31287 FBgn0051287 0 NT CG31453 FBgn0051453 0 TRIP13 thyroid hormone receptor interactor 13 NT CG31716 FBgn0051716 0 NT CG32284 FBgn0052284 0 NT CG3231 FBgn0027522 0 RBBP6 retinoblastoma binding protein 6 NT CG32557 FBgn0052557 0 NT CG32700 FBgn0052700 0 NT CG32727 FBgn0052727 0 DNAJC15 DnaJ (Hsp40) homolog, subfamily C, member 15 NT CG33100 FBgn0053100 0 EIF4E2 eukaryotic translation initiation factor 4E member 2 NT CG3356 FBgn0034989 0 UBE3C ubiquitin protein ligase E3C NT CG3605 FBgn0031493 0 SF382 splicing factor 3b, subunit 2, 145 kDa NT CG3554 FBgn0036004 0 NT CG3700 FBgn0034796 1 TMPRSS9 transmembrane protease, serine 9 NT CG3940 FBgn0037788 0 NT CG4017 FBgn0032143 0 CPB1 carboxypeptidase B1 NT CG4030 FBgn0034585 0 RABEP1 rabaptin, RAB GTPase binding effector protein 1 NT CG4090 FBgn0038492 1 NT CG4291 FBgn0031287 0 WBP4 WW domain binding protein 4 (formin binding protein 21) NT CG4302 FBgn0027073 0 UGT2B10, UGT2B11, UDP glucuronosyltransferase 2 family, UGT2B28 polypeptide B10, B11, B28 NT CG4653 FBgn0030776 0 NT CG4747 FBgn0043456 0 N-PAC cytokine-like nuclear factor n-pac NT CG4851 FBgn0032358 0 PPT2 palmitoyl-protein thioesterase 2 NT CG4901 FBgn0032194 0 DHX33 DEAH (Asp-Glu-Ala-His) box polypeptide 33 NT CG5103 FBgn0036784 0 TKT transketolase (Wernicke-Korsakoff syndrome) NT CG5122 FBgn0032471 0 NT CG5191 FBgn0038803 0 NT CG5567 FBgn0036760 0 LOC283871 hypothetical protein LOC283871 NT CG5715 FBgn0039180 0 NT CG6041 FBgn0029826 1 TMPRSS9 transmembrane protease, serine 9 NT CG6656 FBgn0038912 0 NT CG6717 FBgn0031924 0 SERPIN85 serpin peptidase inhibitor, clade 8 (ovalbumin), member 5 NT CG6763 FBgn0039069 1 NT CG5764 FBgn0037899 0 C15orf15 chromosome 15 open reading frame 15 NT CG6841 FBgn0036828 0 C20orf14 chromosome 20 open reading frame 14 NT CG6906 FBgn0036261 0 NT CG6937 FBgn0038989 0 MKI67IP MKI67 (FHA domain) interacting nucleoter phosphoprotein NT CG7017 FBgn0036951 0 NT CG7290 FBgn0036949 0 NT CG7928 FBgn0039740 0 NT CG8117 FBgn0030683 0 TCEA2 transcription elongation factor A (SII), 2 NT CG9220 FBgn0030662 0 CHSY1 carbohydrate (chondroitin) synthase 1 NT CG9363 FBgn0037697 0 NT CG9520 FBgn0032078 0 C1GALT1 core 1 synthase, glycoprotein-N- acetylgalactosamine 3-beta- galactosyltransferase, 1 NT CG9535 FBgn0027501 0 UAP1 UDP-N-acteylglucosamine pyrophosphorylase 1 NT CG9650 FBgn0029939 2 NT CG9843 FBgn0037237 0 NT CG9947 FBgn0030752 0 TMEM30A transmembrane protein 30A NT comm3 FBgn0053209 0 NT CtBP FBgn0020496 1 CTBP1 C-terminal binding protein 1 NT dbo FBgn0040230 0 KLHL20 kelch-like 20 (Drosophila) NT Dhfr FBgn0004087 0 DHFR dihydrofolate reductase NT dmrt11E FBgn0030477 2 NT drm FBgn0024244 0 NT eas1 FBgn0010110 1 NT ec FBgn0025376 1 NT Ef1alpha100E FBgn0000557 1 EEF1A2 eukaryotic translation elongation factor 1 alpha 2 NT faf FBgn0005632 0 USP9X ubiquitin specific peptidase 9, X-linked (fat facets-like, Drosophila NT fbp FBgn0032820 0 FBP1 fructose-1,6-bisphosphatase 1 NT fred FBgn0051774 0 NT GsD5 FBgn0010041 5 NT GsE2 FBgn0063498 0 NT Hand FBgn0032209 0 HAND2 heart and neural crest derivatives expressed 2 NT HGTX FBgn0040318 0 NKX6-1 NK6 transcription factor related, locus 1 NT Hsp608 FBgn0011244 0 NT i(2)k05713 FBgn0022160 0 GPD2 glycerol-3-phosphate dehydrogenase 2 NT l(3)IX-14 FBgn0002478 0 LMLN leishmanolysin-like (metallopeptidase MB family) NT lota FBgn0005630 2 LOC441636 similar to submaxillary apomucin NT Map60 FBgn0010342 0 NT Mes-4 FBgn0039559 0 WHSC Wolf-Hirschhom syndrome candidate 1 NT Mgat2 FBgn0039738 0 MGAT2 mannosyl (alpha-1,6-)-glycoprotein beta-1,2- N-acetylglucosaminyltransferase NT mol FBgn0028528 0 NIP homolog of Drosophila Numb-interacting protein NT mre11 FBgn0020270 0 MRE11A MRE11 meiotic recombination 11 homolog A NT mRpL15 FBgn0036990 1 MRPL15 mitochondrial ribosomal protein L15 NT mRpL2a FBgn0037833 0 MRPL37 mitochondrial ribosomal protein L37 NT nbs FBgn0026198 1 NBN nibrin NT NfI FBgn0042696 0 NFIA nuclear factor I/A NT nos FBgn0002962 2 NOS1 nitric oxide synthase 1 NT Odc1 FBgn0013307 0 ODC1 ornithine decarboxylase 1 NT Peb FBgn0004181 0 PRB1, PRB2 proline-rich protein BstNI subfamily 1, proline rich protein BstNI subfamily 2 NT PH4alphaEFB FBgn0039776 0 P4HA1 procollagen-proline, 2-oxoglutarate 4- dioxygenase (proline 4-hydoxylase), alpha polypeptide I NT Phax FBgn0033380 0 RNUXA RNA U, small nuclear RNA export adaptor NT ple FBgn0005626 0 TH tyrosine hydroxylase NT Rb97D FBgn0004903 2 LOC144983 heterogeneous nuclear ribonucleoprotein A1 like NT Rbp2 FBgn0010256 0 WBSCR1 Williams-Beuren syndrome chromosome region 1 NT RpI1 FBgn0019938 0 POLR1A polymerase (RNA) I polypeptide A NT RpL10Aa FBgn0038281 0 RPL10A ribosomal protein L10a NT RpS10b FBgn0031035 0 RPS10 ribosomal protein S10 NT Rrp1 FBgn0004584 0 APEX1 APEX nuclease (multifunctional DNA repair enzyme) 1 NT salr FBgn0000287 0 SALL3 sal-like 3 NT sda FBgn0015541 1 ARTS-1 type 1 tumor necrosis factor receptor shedding aminopeptidase regulator NT SF1 FBgn0025571 0 SF1 splicing factor 1 NT shn FBgn0003396 0 NT Sirt2 FBgn0038788 0 SIRT2 sirtuin (silent mating type information regulation 2 homolog) 2 NT snRNP69D FBgn0016940 0 SNRPD1 small nuclear ribonucleoprotein D1 polypeptide 16 kDa NT Spn43Ab FBgn0024293 0 NT Spt3 FBgn0037981 1 NT sqd FBgn0003498 0 NT ST6Gal FBgn0035050 0 ST6GAL2 ST6 beta-galactosamide alpha-2,6- sialyltranferase 2 NT stau FBgn0003520 0 STAU staufen, RNA binding protein NT stich1 FBgn0016941 1 NT svr FBgn0004648 0 CPD carboxypeptidase D NT T3dh FBgn0017482 1 ADHFE1 alcohol dehydrogenase, iron containing, 1 NT Tdp1 FBgn0051953 0 TDP1 tyrosyl-DNA phosphodiesterase 1 NT tth FBgn0030502 5 NT Ugt86Dd FBgn0040256 0 NOVEL 5 CG17142 FBgn0035112 0 4 CG14076 FBgn0036829 0 4 CG14870 FBgn0038342 0 EPPB9 B9 protein 4 CG31145 FBgn0051145 0 FAM20C family with sequence similarity 20, member C 4 CG31203 FBgn0051203 0 4 CG31288 FBgn0051288 0 4 CG4585 FBgn0025335 0 4 CG7706 FBgn0038640 0 SLC4A1AP solute carrier family 4 (anion exchanger), member 1, adaptor protein 4 Osi10 FBgn0037417 0 3 CG14084 FBgn0036855 0 3 CG14556 FBgn0039413 0 3 CG14744 FBgn0033324 0 SLC24A6 solute carrier family 24 (sodium/potassium/calcium exchanger), member 6 3 CG14945 FBgn0032402 0 3 CG17005 FBgn0032109 0 3 CG1968 FBgn0033401 0 COG6 component of oligomeric golgi complex 6 3 CG1971 FBgn0039881 0 3 CG3566 FBgn0029854 0 CYB5-M outer mitochondrial membrane cytochrome b5 3 CG4786 FBgn0037012 0 3 CG8740 FBgn0027585 0 3 CG9264 FBgn0032911 0 3 CG9525 FBgn0032080 0 2 CG10946 FBgn0029974 0 2 CG1113 FBgn0037304 0 2 CG11381 FBgn0029568 3 2 CG12688 FBgn0029707 0 2 CG12958 FBgn0034018 0 2 CG14314 FBgn0038581 0 2 CG14354 FBgn0039376 0 2 CG15897 FBgn0029857 0 WDR4 WD repeat domain 4 2 CG16786 FBgn0034974 0 2 CG30389 FBgn0050389 0 TMEM57 transmembrane protein 57 2 CG32224 FBgn0036950 0 2 CG3704 FBgn0040346 0 XAB1 XPA binding protein 1, GTPase 2 CG4098 FBgn0036648 0 NUDT9 nudix (nucleoside diphosphate linked moiety X)-type motif 9 2 CG4643 FBgn0043010 0 FBXO45 F-box protein 45 2 CG5308 FBgn0037908 3 2 CG5348 FBgn0034156 0 SLC24A6 solute carrier family 24 (sodium/potassium/calcium exchanger), member 6 2 CG9205 FBgn0035181 0 2 CG9752 FBgn0034614 0 C9orf64 chromosome 9 open reading frame 64 2 nes FBgn0026630 0 C3F putative protein similar to nessy 1 CG10514 FBgn0039312 0 1 CG13659 FBgn0039319 0 1 CG14160 FBgn0036066 0 SLC2A5 solute carrier family 2 (facilitated glucose/fructose transporter), member 5 1 CG14515 FBgn0039648 0 1 CG14629 FBgn0040398 1 1 CG14743 FBgn0033326 0 SLC24A6 solute carrier family 24 (sodium/potassium/calcium exchanger), member 6 1 CG18679 FBgn0040663 0 1 CG2879 FBgn0025834 0 LRRC8B leucine rich repeat containing 8 family, member B 1 CG2921 FBgn0034689 1 1 CG3106 FBgn0030148 0 1 CG31410 FBgn0051410 0 1 CG32159 FBgn0052159 0 1 CG32637 FBgn0052637 0 LGR8 leucine-rich repeat-containing G protein- coupled receptor 8 1 CG3634 FBgn0037026 0 ST7 suppression of tumorigenicity 7 1 CG8858 FBgn0033698 0 KIAA0368 KIAA0368 1 mars FBgn0033845 0 DLG7 discs, large homolog 7 1 Osi16 FBgn0051561 0 1 sip2 FBgn0031878 0 0 CG10095 FBgn0037993 2 0 CG10183 FBgn0039093 2 0 CG13188 FBgn0033668 8 0 CG14162 FBgn0040823 0 0 CG14471 FBgn0033049 0 0 CG2185 FBgn0037358 0 CHP calcium binding protein P22 0 CG2656 FBgn0037478 0 ATPBD1C ATP binding domain 1 family, member C 0 CG31189 FBgn0051189 0 0 CG32432 FBgn0052432 0 0 CG3536 FBgn0050267 0 CNGA1 cyclic nucleotide gated channel alpha 1 0 I(1)G0331 FBgn0029944 8 0 Osi18 FBgn0037428 0 0 ppk13 FBgn0032912 0 NT CG10200 FBgn0033968 0 NT CG10424 FBgn0036848 0 FLJ10769 hypothetical protein FLJ10769 NT CG10589 FBgn0037035 0 NT CG11073 FBgn0034693 0 NT CG11113 FBgn0033165 0 NT CG11310 FBgn0037067 0 NT CG11576 FBgn0039882 0 C20orf54 chromosome 20 open reading frame 54 NT CG11634 FBgn0032968 0 NT CG11672 FBgn0037563 0 NT CG11699 FBgn0030311 0 NT CG11750 FBgn0030294 0 NT CG11839 FBgn0039271 0 CCDC16 coiled-coil domain containing 16 NT CG11847 FBgn0039281 0 SDCCAG1 serologically defined colon cancer antigen 1 NT CG11875 FBgn0039301 0 NUP37 nucleoporin 37 kDa NT CG11881 FBgn0039638 0 NT CG11926 FBgn0031640 0 MON1A MON1 homolog A NT CG12508 FBgn0040995 0 NT CG12584 FBgn0037257 0 NT CG12608 FBgn0030630 1 PAK1IP1 PAK1 interacting protein 1 NT CG12672 FBgn0030886 1 NT CG12985 FBgn0030881 0 RDBP RD RNA binding protein NT CG13014 FBgn0030759 1 NT CG13021 FBgn0029669 0 NT CG13075 FBgn0036563 0 NT CG13086 FBgn0032770 0 NT CG13088 FBgn0032047 0 PGDS prostaglandin D2 synthase, hematopoietic NT CG13169 FBgn0033704 0 NT CG13239 FBgn0037197 0 NT CG13364 FBgn0026879 0 HSPC016 hypothetical protein HSPC016 NT CG13538 FBgn0034820 0 NT CG13552 FBgn0034864 0 NT CG13599 FBgn0039128 0 NT CG13615 FBgn0039199 2 NT CG13623 FBgn0039205 0 NT CG13654 FBgn0039290 0 NT CG13785 FBgn0031901 0 NT CG13836 FBgn0039060 0 NT CG1394 FBgn0030277 9 NT CG13984 FBgn0031796 0 NT CG14017 FBgn0031721 0 MGC35043 hypothetical protein MGC35043 NT CG14047 FBgn0040390 0 NT CG14082 FBgn0036851 1 NT CG14131 FBgn0036205 0 NT CG14252 FBgn0039462 0 NT CG14423 FBgn0029646 3 NT CG14448 FBgn0037191 0 NT CG14453 FBgn0037179 2 NT CG14550 FBgn0039405 1 DSCR5 Down syndrome critical region gene 5 NT CG14563 FBgn0037139 0 NT CG14564 FBgn0037131 0 NT CG14565 FBgn0037129 0 NT CG14574 FBgn0037104 0 NT CG14609 FBgn0037483 0 KIAA1212 KIAA1212 NT CG14659 FBgn0037284 0 NT CG14662 FBgn0037291 0 NT CG14843 FBgn0038230 0 NT CG14850 FBgn0038239 0 NT CG14931 FBgn0032374 0 NT CG15059 FBgn0030905 0 NT CG15133 FBgn0032619 0 NT CG15152 FBgn0032665 0 NT CG15278 FBgn0032554 0 NT CG1529 FBgn0031144 1 ZNF569 zinc finger protein 569 NT CG15366 FBgn0030080 0 NT CG15376 FBgn0029692 5 NT CG15432 FBgn0031603 2 NT CG15471 FBgn0029726 0 NT CG15488 FBgn0032440 0 NT CG15513 FBgn0039705 0 ATG16L ATG16 autophagy related 16-like NT CG15771 FBgn0029801 0 HDHD4 haloacid dehalogenase-like hydrolase domain containing 4 NT CG15784 FBgn0029766 1 NT CG15888 FBgn0038131 0 NT CG1678 FBgn0031176 0 NT CG16865 FBgn0028919 0 FLJ22965 hypothetical protein FLJ22965 NT CG16964 FBgn0032385 0 NT CG17261 FBgn0031501 0 NT CG17267 FBgn0038821 0 NT CG17282 FBgn0038857 0 NT CG17382 FBgn0039080 0 NT CG17786 FBgn0039167 1 CNOT6 CCR4-NOT transcription complex, subunit 6 NT CG17807 FBgn0034748 0 LOC91801 hypothetical protein BC015183 NT CG17952 FBgn0034657 0 NT CG18145 FBgn0032189 0 NT CG18275 FBgn0029523 2 NT CG18368 FBgn0033864 0 NT CG18600 FBgn0038601 0 NT CG1896 FBgn0039870 0 NT CG2016 FBgn0037289 0 NT CG2124 FBgn0030217 0 FLJ13149 hypothetical protein FLJ13149 NT CG2889 FBgn0030206 0 NT CG30010 FBgn0050010 0 MGC70857 similar to RIKEN cDNA C030006K11 gene NT CG30101 FBgn0050101 1 NT CG30109 FBgn0050109 0 P53CSV p53-inducible cell-survival factor NT CG30363 FBgn0050363 0 NT CG30419 FBgn0050419 0 NT CG31093 FBgn0051093 0 NT CG31389 FBgn0051389 0 NT CG31407 FBgn0051407 0 NT CG31825 FBgn0051825 1 NT CG31989 FBgn0051989 0 NT CG31998 FBgn0051998 0 NT CG32021 FBgn0052021 1 NT CG32345 FBgn0052345 3 NT CG32436 FBgn0052436 0 NT CG32639 FBgn0052639 0 NT CG32783 FBgn0029686 0 NT CG33109 FBgn0053109 0 NT CG33267 FBgn0053267 2 NT CG3330 FBgn0039511 0 NT CG33301 FBgn0053301 3 NT CG33340 FBgn0053340 0 NT CG3406 FBgn0036008 0 PRO1855 hypothetical protein PRO1855 NT CG3501 FBgn0034791 0 C14orf122 chromosome 14 open reading frame 122 NT CG3546 FBgn0029716 4 NT CG3596 FBgn0025645 0 NT CG3713 FBgn0040343 0 NT CG3764 FBgn0036684 0 NT CG3800 FBgn0034802 0 ZNF9 zinc finger protein 9 (a cellular retroviral nucleic acid binding protein) NT CG3805 FBgn0031665 0 NT CG3973 FBgn0029881 0 NT CG40402 FBgn0058402 0 NT CG4627 FBgn0033808 0 C16orf51 chromosome 16 open reading frame 51 NT CG4820 FBgn0037876 0 ZNF136 zinc finger protein 136 NT CG5237 FBgn0038593 1 KIAA1409 KIAA1409 NT CG5323 FBgn0034362 0 NT CG5386 FBgn0038945 10 NT CG5467 FBgn0039433 3 NT CG5468 FBgn0039434 0 NT CG5538 FBgn0038052 0 NT CG5955 FBgn0035997 0 NT CG6018 FBgn0034736 0 NT CG6073 FBgn0039417 0 LOC51236 brain protein 16 NT CG6195 FBgn0038723 1 DRG2 developmentally regulated GTP binding protein 2 NT CG6301 FBgn0034161 0 NT CG6480 FBgn0036964 0 FRG1 FSHD region gene 1 NT CG6569 FBgn0038909 0 MYH2 myosin, heavy polypeptide 2, skeletal muscle, adult NT CG6614 FBgn0032369 0 TTC18 tetratricopeptide repeat domain 18 NT CG6631 FBgn0039206 0 NT CG7053 FBgn0030960 0 FLJ11773 hypothetical protein FLJ11773 NT CG7200 FBgn0032671 1 JMJD4 jumonji domain containing 4 NT CG7242 FBgn0040494 0 NT CG7381 FBgn0038098 0 NT CG7567 FBgn0039670 0 NT CG8031 FBgn0038110 0 C2orf4 chromosome 2 open reading frame 4 NT CG8420 FBgn0037664 0 NT CG8538 FBgn0038223 0 NT CG8852 FBgn0031548 1 LRRTM4 leucine rich repeat transmembrane neuronal 4 NT CG9328 FBgn0032886 0 NT CG9380 FBgn0035094 0 NT CG9773 FBgn0037609 0 NT CR32205 FBgn0052205 1 NT Edg78E FBgn0000551 0 NT I(1)G0196 FBgn0027279 0 KIAA0433 KIAA0433 protein NT I(1)G0222 FBgn0028343 0 NT Mkm1 FBgn0029152 1 MKRN1 makorin, ring finger protein, 1 NT msb1l FBgn0027949 0 NT MTA1-like FBgn0027951 4 MTA1 metastasis associated 1 NT nito FBgn0027548 0 RBM15 RNA binding motif protein 15 NT olf186-M FBgn0015522 0 NT Osi13 FBgn0037422 0 NT Osi17 FBgn0037427 0 NT Osi19 FBgn0037429 0 NT Pcp FBgn0003046 0 NT sano FBgn0034408 0 NT T48 FBgn0004359 0 NT yellow-d2 FBgn0034856 0

TABLE II Inhibition of NFAT nuclear localization # of Identity of in TG- potential potential off- treated off-targets targets of Gene Description CG Amplicon No. Expression cells of 21 nt 21 nt CanA1 Calcineurin A1 CG1455 DRSC16600 +/− − 0 Pp2B-14D Protein phosphatase CG9842 DRSC23315 ++ +++ 0 2B at 14D DRSC20270 +++ 1 CG12238 CanA-14F Calcineurin A at 14F CG9619 DRSC23296 + +++ 0 DRSC20211 +++ 13 not listed CanB Calcineurin B CG4209 DRSC18449 +/− ++ 0 CanB2 Calcineurin B2 CG11217 DRSC07355 ++ ++ 0 CG32812 CG32812 CG32812 DRSC18478 + − 0 # of Identity of # of potential potential off- potential Identity of off-targets targets of off-targets potential off- Gene of 20 nt 20 nt of 19 nt targets of 19 nt Comments CanA1 0 1 CG7952 Pp2B-14D 0 0 2 CG12238, 3 CG12238, CG32223 CG32223, CG32025 CanA-14F 0 0 56 not listed 163 not listed CG9642 (Pp2B-14D) has 18 matches with this amplicon. CanB 1 CG11217 2 CG11217 (CanB2), (CanB2) CG15859 CanB2 1 CG4209 2 CG4209 (CanB), (CanB) CG5744 CG32812 0 0

TABLE III Potential off-target Description of the potential off-target (NCBI Gene) Molecule in Suppl Table III I. DIRECT NFAT KINASES Shaggy CG13772 neurexin binding; ectoderm development and neurogenesis; (sgg. CG2621) (neuroligin) CG4771 NA CG12199 peroxidase activity, cell adhesion, defense response; reactive oxygen species metabolism; transmission of nerve (kek5) impulse; CG1049 choline-phosphate cytidylyltransferase activity; (cct1) CG5907 calcium sensitive guanylate cyclase activator activity; calmodulin binding; neurotransmitter secretion; synaptic (frq) transmission; CG32538 nicotinic acetylcholine-activated cation-selective channel activity; muscle contraction; nerve-nerve synaptic (nAcRalpha- 18C) CG9176 intracellular cyclic nucleotide activated cation channel activity; potassium channel activity; sensory perception; (cng1) signal transduction; CG3427 cAMP-dependent protein kinase regulator activity; small GTPase mediated signal transduction; (epec) CG33513 N-methyl-D-aspartate selective glutamate receptor activity; cation transport; nerve-nerve synaptic transmission; (nmdar2) CG13290 NA CG12708 NA CG4136 nucleobase, nucleoside, nucleotide and nucleic acid metabolism; regulation of transcription from RNA polymerase II promoter; ligand-dependent nuclear receptor activity; Gasket CG12212 transcription factor activity; leading edge cell fate determination; ectoderm development; photoreceptor cell (gsk. CG11338, (peb) morphogenesis; maintenance of tracheal epithelial integrity; negative regulation of JNK cascade; CG31003) CG12147 CG6205 acyltransferase activity; cell adhesion; regulation of Wnt receptor signaling pathway; (por) CG14895 receptor signaling protein serine/threonine kinase activity; MAPKKK cascade; actin filament organization; cell (pak3) proliferation; cytoskeleton organization and biogenesis; CG18214 Rho guanyl-nucleotide exchange factor activity; actin cytoskeleton organization and biogenesis; axon guidance; (trio) central and peripheral nervous system development; transmission of nerve impulse. Disc overgrown CG2028 receptor signaling protein serine/threonine kinase activity; Wnt receptor signaling pathway; negative regulation (den. CG2048) smoothened signaling pathway; regulation of proteolysis and peptidolysis; CK1 alpha CG2048 receptor signaling protein serine/threonine kinase activity; Wnt receptor signaling pathway; negative regulation (CG2028) (ckIalpha) smoothened signaling pathway; regulation of proteolysis and peptidolysis; CG2577 receptor signaling protein serine/threonine kinase activity; casein kinase I activity; CG9102 transcription factor activity; chromatin assembly or disassembly; eye-antennal disc metamorphosis; sex (bab2) determination; female gonad development; leg morphogenesis; transmission of nerve impulse. CG7838 receptor signaling protein serine/threonine kinase activity; chromosome segregation; mitotic spindle checkpoint (bub1) regulation of exit from mitosis. CG7892 receptor signaling protein serine/threonine kinase activity; anti-apoptosis; cell proliferation; establishment of (nmo) planar polarity; eye morphogenesis; wing morphogenesis; negative regulation of Wnt receptor signaling pathway; negative regulation of frizzled signaling pathway; CG16973 JUN kinase kinase kinase kinase activity; small GTPase regulator activity; oogenesis; photoreceptor cell (msn) morphogenesis; regulation of cell shape; CG2577 CG2048 receptor signaling protein serine/threonine kinase activity; casein kinase I activity; cell communication; circadian rhythm; imaginal disc growth; regulation of ecdysteroid secretion; regulation of protein-nucleus import; CG2028 receptor signaling protein serine/threonine kinase activity; Wnt receptor signaling pathway; negative regulation smoothened signaling pathway; regulation of proteolysis and peptidolysis; CG7838 receptor signaling protein serine/threonine kinase activity; chromosome segregation; mitotic spindle checkpoint; (bub1) regulation of exit from mitosis CG7236 receptor signaling protein serine/threonine kinase activity; cytokinesis; regulation of progression through cell cycle; CG3228 ATP-dependent helicase activity; nuclear mRNA splicing, via spliceosome; proteolysis and peptidolysis. (kurz) CG7094 CG9135 guanyl-nucleotide exchange factor activity; proteolysis and peptidolysis. CG9962 CG5621 glutamate-gated ion channel activity; kainate selective glutamate receptor activity; potassium channel activity; nerve-nerve synaptic transmission. II. OTHER KINASES CG31640 CG33531 transmembrane receptor protein tyrosine kinase activity; cell-cell adhesion; ectoderm development; mesoderm (ddr) development; nervous system development; CG2699 phosphoinositide 3-kinase regulator activity; insulin receptor signaling pathway; positive regulation of cell size (Pi3K21B) positive regulation of growth; regulation of cell proliferation; regulation of cell size; Pelle CG5263 mRNA 3′-UTR binding; translation repressor activity; (pil. CG5974) I(1)G0148 CG9463 alpha-mannosidase activity; hydrolase activity, hydrolyzing N-glycosyl compounds. (CG32742) Pole hole CG8522 fatty acid biosynthesis; positive regulation of transcription; transcription from RNA polymerase II promoter; (phl. CG2845) (HLH106) CG11073 NA CG3634 NA CG15105 transcription regulator activity; ubiquitin-protein ligase activity; CG3198 nuclear mRNA splicing, via spliceosome CG17299 receptor signaling protein serine/threonine kinase activity; defense response; fatty acid metabolism; regulation phosphate metabolism; response to stress CG8465 NA Foraging CG7826 receptor signaling protein serine/threonine kinase activity; nervous system development; ectoderm development; (for. CG10033) (mnb) olfactory learning; cell proliferation; circadian rhythm; induction of apoptosis; learning and/or memory; CG32629 NA CG13472 NA CG18389 transcription factor activity; autophagy; ecdysone-mediated induction of salivary gland cell death; induction of (Elp93F) apoptosis by hormones; Iarval midgut histolysis; CG9310 steroid hormone receptor activity; regulation of transcription from RNA polymerase II promoter; endoderm (hnf4) development; mesoderm development; CG16902 steroid hormone receptor activity; metamorphosis; regulation of transcription from RNA polymerase II (Hr4) promoter CG4013 corepressor activity; regulation of transcription from RNA polymerase II promoter. (smr) CG8949 NA CG14447 glutamate receptor binding; determination of muscle attachment site; (grip) CG5683 RNA polymerase II transcription factor activity; cell proliferation; (Aef1) CG32180 specific RNA polymerase II transcription factor activity; autophagy; cell death; salivary gland cell death (elp74EF) mesoderm development; oogenesis; CG32423 mRNA processing; CG3696 ATP-dependent helicase activity; blastoderm segmentation; chromatin assembly or disassembly; (kis) CG3695 RNA polymerase II transcription mediator activity; mediator complex; (MED23) CG14023 NA CG13109 transcription coactivator activity; signal transducer activity; border follicle cell migration; (tal) CG9381 learning and/or memory; olfactory learning; (mura) CG5466 NA CG12254 RNA polymerase II transcription mediator activity; (MED25) CG9354 nucleic acid binding; structural constituent of ribosome; (RpL34b) CG6575 carbohydrate binding; cell adhesion; heterophilic cell adhesion; nervous system development. (glec) CG14366 NA CG1161 NA CG10732 NA CG7368 NA CG12432 NA CG17888 transcription factor activity; circadian rhythm; mesoderm development; (Pdp1) PI3K59F CG3856 octopamine receptor activity; octopamine/tyramine signaling pathway; ovulation; (CG5373) (Oamb) CG14619 cysteine-type endopeptidase activity; ubiquitin thiolesterase activity; ubiquitin-specific protease activity CG10989 NA III. OTHER CG6919 CG18208 G-protein coupled receptor protein signaling pathway; transmission of nerve impulse. CG31288 CG15415 NA CG32381 neurotransmitter secretion; synaptic vesicle priming. (une-13-4A) Molecule in Suppl Table II CanA1 CG7952 (giant) negative regulation of transcription from RNA polymerase II promoter; posterior head segmentation; (CG1455) terminal region determination; zygotic determination of anterior/posterior axis; ring gland development; salivary gland development; torso signaling pathway. Pp2B-14D CG12238 chromatin binding; transcription regulator activity; gene silencing; oogenesis. (CG98-42) (1(1)G0084) CG32223 NA CG32025 NA CanA-14F not listed (CG9819) CanB CG11217 (CanB2) calcium-dependent protein serine/threonine phosphatase activity; cell homeostasis; neurotransmitter (CG4209) secretion; vesicle-mediated transport. CG15859 NA CG11217 CG4209 (CanB) calcium-dependent protein serine/threonine phosphatase activity; cell homeostasis; neurotransmitter (CanB2) secretion; vesicle-mediated transport. CG5744 calcium-mediated signaling; sensory perception; signal transduction; visual perception.

TABLE IV # of Score in Description potential primary of the human Amplicon off-targets Identity of potential screen Gene homologus CG FBgn No. of 21 nt off-targets of 21 nt L DIRECT NFAT KINASES QSK3 6 shsggy (sgg) GSK3B CG2621 FBgn0003371 DRSC18832 4 CG5907, CG13772, CG12199, CG1049 1 gssket (gskt) GSK3A CG11338, FBgn0046332 DRSC14058 0 CG31003 CK1 4 gilgamesh (gtsh) CSNK1G CG8963 FBgn0011253 DRSC16154 0 3 CG12147 CSNK1 CG12147 FBgn0037325 DRSC12192 0 1 dscs overgrown CSNK1E CG2048 FBgn0002413 DRSC16929 0 (dco) 1 CK1alpha CSNK1A1 CG2028 FBgn0015024 DRSC20231 3 CG2048, CG2577, CG7836 1 CG2577 CSNK1 CG2577 FBgn0030364 DRSC19663 3 CG2028, CG2048, CG7838 1 CG7094 CSNK1 CG7094 FBgn0032650 DRSC03005 0 0 CG9962 CSNK1 CG9962 FBgn0031441 DRSC00739 0 DYRK 1 CG40478 DYRKZ CG40476 FBgn0009975 DRSC21055 0 L OTHER KINASES DDR 4 CG31640 DDR CG31640 FBgn0051640 DRSC2504 0 IRAK 3 pI (pI (pala) IRAK CG5974 FBgn0010441 DRSC17026 0 CK2 3 Ck1alpha CSNK2A CG17520 FBgn0000256 DRSC11945 0 CDC7 2 I(I)GO148 CDC7 CG32742 FBgn0026360 DRSC18429 0 TRRAP 2 Nipped-A TRRAP CG2905, FBgn0004661, DRSC4882 0 CG33554 FBgn0053554, FBgn0039969 RAF 1 phl (pola hols) RAF CG2845 FBgn0003079 DRSC18821 2 CG11073, CG8522 PRKG1 1 for (for aging) PRKG1 CG10033 FBgn0000721 DRSC00195 2 CG32629, CG18389 PI3K 1 PI3K59F PIK3C3 CG5373 FBgn0015277 DRSC04840 0 L OTHER HTR 5 CG6919 HTR4 CG5919 FBgn0038980 DRSC16134 0 FAM20 4 CG31145 FAM20C CG31145 FBgn0051145 DRSC14671 0 4 CG31288 CG31288 FBgn0051288 DRSC14667 0 B9 4 CG14870 EPPB9 CG14870 FBgn0038342 DRSC14993 0 4 CG4585 CG4585 FBgn0025335 DRSC4475 0 PGLYRP 3 CG8995 PGLYRP3 CG8995 FBgn0030695 DRSC20137 0 STM 3 Stm STM1 CG9129 FBgn0045073 DRSC20158 0 Cathepsin 8 2 CG3074 CTS8 CG3074 FBgn0034709 DRSC4334 0 # of # of Score in potential potential primary off-targets Identity of potential off-targets screen of 20 nt off-targets of 20 nt of 19 nt Identity of potential off-targets of 19 nt L DIRECT NFAT KINASES QSK3 6 7 CG5907, CG13772, 12 CG4771, CG5907, CG13772, CG12199, CG12199, CG1049, CG1049, CG32538, CG9178, CG3427, CG32538, CG9178, CG32513, CG13290, CG12708, CG3427 CG4136 1 0 1 CG12212 CK1 4 0 0 3 2 CG6205, CG14895 3 CG6205, CG14895, CG18214 1 1 CG2028 1 CG2028 1 4 CG2048, CG2577, 6 CG2048, CG2577, CG7836, CG18973, CG7836, CG18973 CG7892, CG9102 1 4 CG2028, CG2048, 5 CG2028, CG2048, CG7838, CG7236, CG7838, CG7236 CG3228 1 1 CG9135 1 CG9135 0 0 1 CG5621 DYRK 1 0 0 L OTHER KINASES DDR 4 2 CG33531, CG2699 2 CG33531, CG2699 IRAK 3 1 CG5263 1 CG5263 CK2 3 0 0 CDC7 2 1 CG9483 1 CG9483 TRRAP 2 0 0 RAF 1 4 CG11073, 7 CG3196, CG11073, CG8522, CG3834, CG8522, CG3634, CG15105, CG17299, CG8485 CG15105 PRKG1 1 4 CG32629, CG18389, 27 CG7825, CG9310 PI3K 1 2 CG14619, CG10969 3 CG14619, CG3856, CG10969 L OTHER HTR 5 0 1 CG16205 FAM20 4 0 0 4 1 CG15415 2 CG15415, CG32381 B9 4 0 0 4 0 0 PGLYRP 3 0 0 STM 3 0 0 Cathepsin 8 2 0 0 

1. A method of identifying an agent that modulates an NFAT regulator protein, comprising: contacting at least one test agent with a recombinant cell comprising at least one NFAT regulator protein or fragment or derivative thereof; assessing the effect of the test agent on an activity, interaction, expression, or binding to the NFAT regulator protein or fragment or derivative thereof; and identifying the test agent that has an effect on an activity, interaction, expression, or binding to the NFAT regulator protein or fragment or derivative thereof, whereby the identified test agent is characterized as an agent that modulates an NFAT regulator protein.
 2. The method of claim 1, wherein the NFAT regulator protein is encoded by at least one NFAT regulator selected from the group consisting of ORAI1 (SEQ ID NO:1), ORAI2 (SEQ ID NO:2), ORAI3 (SEQ ID NO:3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) and DYRK6 (SEQ ID NO:9).
 3. The method of claim 1, wherein the NFAT regulator protein is encoded by at least one of the genes listed in Table I.
 4. The method of claim 2, wherein assessing the effect of the test agent comprises using an antibody which specifically binds to a NFAT regulator protein encoded by ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8), or DYRK6 (SEQ ID NO:9).
 5. The method of claim 1, further comprising assessing the effect of the test agent on electrical current across the plasma membrane of the cell.
 6. The method of claim 5, wherein the electrical current is due to flux of monovalent cations or divalent cations across the cell.
 7. The method of claim 1, further comprising assessing the effect of the test agent on intracellular calcium within the cell.
 8. The method of claim 7, further comprising identifying the test agent that has an effect on intracellular calcium within the cell, whereby the identified test agent is characterized as an agent that modulates intracellular calcium and an agent that modulates NFAT regulator protein.
 9. The method of claim 1, wherein the cell comprises at least one heterologous NFAT regulator proteins or a fragment or derivative thereof.
 10. The method of claim 1, wherein the cell comprises heterologous nucleic acid encoding at least one NFAT regulator protein or a fragment or derivative thereof.
 11. The method of claim 1, wherein the cell overexpresses or underexpresses at least one NFAT regulator protein or fragment or derivative thereof.
 12. A method of identifying an agent that modulates intracellular calcium, comprising: contacting at least one test agent with a recombinant cell comprising at least one NFAT regulator protein or fragment or derivative thereof; assessing the effect(s) of the test agent on intracellular amounts or concentrations of cations or divalent cations within the cell, or on ion influx into the cell; and identifying the test agent that has an effect on intracellular amounts or concentrations of cations or divalent cations within the cell, or on ion influx into the cells, whereby the identified test agent is characterized as an agent that modulates intracellular calcium.
 13. The method of claim 12, wherein the intracellular cation is calcium.
 14. The method of claim 13, wherein assessing the effect of the test agent comprises monitoring calcium levels in the cytoplasm, monitoring calcium levels in an intracellular calcium store, monitoring calcium movement, or monitoring a calcium-entry mediated event.
 15. The method of claim 12, further comprising assessing the effect of the test agent on an activity, interaction, expression, or binding to the NFAT regulator protein or fragment or derivative thereof.
 16. The method of claim 15, wherein the NFAT regulator protein is encoded by at least one NFAT regulator selected from the group consisting of ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) or DYRK6 (SEQ ID NO:9).
 17. The method of claim 15, wherein the agent that modulates intracellular calcium is further characterized as an agent that modulates NFAT regulator protein.
 18. The method of claim 12, wherein the recombinant cell comprises at least one heterologous NFAT regulator protein or a fragment or derivative thereof.
 19. The method of claim 12, wherein the recombinant cell comprises heterologous nucleic acid encoding at least one NFAT regulator protein or a fragment or derivative thereof.
 20. The method of claim 12, wherein the recombinant cell overexpresses at least one NFAT regulator protein or fragment or derivative thereof.
 21. The method of claim 12, wherein the recombinant cell exhibits dyshomeostasis.
 22. The method of claim 12, wherein the recombinant cell exhibits calcium dyshomeostasis
 23. A method to screen for an agent that modulates NFAT regulator function, comprising: administering at least one test agent to a cell comprising at least one vector that comprises a heterologous nucleic acid encoding at least one NFAT regulator protein, or a fragment or derivative thereof, and at least one NFAT regulatory domain, fragment, or derivative thereof, that is operably linked to a reporter protein; and monitoring intracellular localization of at least one expression product encoded by the vector, whereby the test agent that has an effect on intracellular localization of the expression product is characterized as an agent that modulates NFAT regulator function.
 24. The method of claim 23, wherein the agent that modulates NFAT regulator function is associated with cytoplasmic or nuclear localization of the expression product.
 25. The method of claim 23, wherein the cell is under resting conditions.
 26. The method claim 23, wherein the cell is stimulated with a calcium modulating agent.
 27. The method of claim 26, wherein the cell is stimulated with thapsigargin or ionomycin.
 28. A method to diagnose unexplained immunodeficiency in a subject comprising: sequencing at least 25 contiguous nucleotides in a gene from the subject corresponding to ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8), DYRK6 (SEQ ID NO:9), or any of the genes listed in Table I; and comparing the sequence of the subject's gene to the wild type sequence of the gene, wherein an variation between the gene from the wild type sequence indicates the subject's gene is responsible for the immunodeficiency.
 29. The method of claim 28, wherein the comparison comprises obtaining a biological sample from the subject, sequencing the DNA in the biological sample, and electronically aligning the DNA sequence obtained from the biological sample to a wild type sequence.
 30. The method of claim 28, wherein the variation comprises a nucleotide mutation from C to T at position 271 of the coding sequence of ORAI1 (SEQ ID NO: 1).
 31. The method of claim 28, wherein the unexplained immunodeficiency is associated with defects in regulation of NFAT activity.
 32. The method of claim 28, wherein the variation comprises a mutation in a splice site.
 33. The method of claim 28, wherein the variation comprises a nonsynonymous mutation.
 34. A method for identifying an agent for treating or preventing a disease or disorder associated with a NFAT regulator protein, comprising: assessing the effects of a test agent on an organism exhibiting a disease or disorder associated with NFAT regulator protein and identifying the test agent as an agent for treating or preventing a disease or disorder associated with NFAT regulator protein if it has an effect on a phenotype of the organism associated with the disease or disorder, wherein the test agent modulates an activity, interaction, expression, or binding of at least one NFAT regulator protein or fragment or derivative thereof.
 35. The method of claim 34, wherein the organism comprises one or more cells that exhibit calcium dyshomeostasis.
 36. The method of claim 34, wherein the disease or disorder associated with NFAT regulator protein is rheumatoid arthritis, inflammatory bowel disease, allogeneic or xenogeneic transplantation rejection, graft-versus-host disease, aplastic anemia, psoriasis, lupus erytematosus, inflammatory disease, MS, type I diabetes, asthma, pulmonary fibrosis, scleroderma, dermatomyositis, Sjogren's syndrome, postpericardiotomy syndrome, Kawasaki disease, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pemphigus vulgaris, autoimmune hemolytic anemia, idiopathic thrombopenia, chronic glomerulonephritis, Goodpasture's syndrome, Wegner's granulomatosis, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, uveitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's disease, ulcerative colitis, colitis/inflammatory bowel syndrome, Guilllain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, eczema, and autoimmune thyroiditis. Transplant graft rejections, acquired immunodeficiencies, common variable immunodeficiency, myocardial hypertrophy, severe combined immunodeficiency, dilated cardiomyopathy, excessive or pathological bone resorption, excessive adipocyte differentiation, obesity, or reactivation of latent viruses.
 37. An antibody which specifically binds to a NFAT regulator protein encoded by ORAI1 (SEQ ID NO: 1), ORAI2 (SEQ ID NO: 2), or ORAI3 (SEQ ID NO: 3), DYRK1A (SEQ ID NO:4), DYRK1B (SEQ ID NO:5), DYRK2 (SEQ ID NO:6), DYRK3 (SEQ ID NO:7), DYRK4 (SEQ ID NO:8) or DYRK6 (SEQ ID NO:9).
 38. The antibody of claim 37, wherein the NFAT regulator protein is produced by means of culturing a host cell comprising a recombinant vector, the recombinant vector comprising a nucleic acid sequence, the nucleic acid sequence encoding the NFAT regulator or fragment or derivative thereof, wherein the host cell is cultured under conditions suitable for expression of the NFAT regulator.
 39. A system comprising: an isolated cell comprising at least one heterologous NFAT regulator protein or fragment or derivative thereof, and/or at least one heterologous nucleic acid encoding a NFAT regulator protein or fragment or derivative thereof; and a monitoring agent used to monitor, detect, or measure electrical current across the plasma membrane of the cell.
 40. The system of claim 39, wherein the electrical current is due to flux of cations or divalent ions across the cell.
 41. The system of claim 40, wherein the monitoring agent is used to monitor the effect of a test agent on intracellular calcium within the cell.
 42. The system of claim 40, wherein the monitoring agent is used to monitor, detect, or measure a calcium-entry mediated event.
 43. A system comprising: a recombinant cell overexpressing at least one mammalian NFAT regulator protein or fragment or derivative thereof; and a monitoring agent used to monitor, detect, or measure a calcium-entry mediated event.
 44. The system of claim 43, wherein the NFAT regulator is encoded by at least one nucleic acid selected from the group consisting of ORAI1 (NM_(—)032790), ORAI2 (BC069270), ORAI3 (NM_(—)152288), DYRK1A (NM_(—)001396), DYRK1B (NM_(—)004714), DYRK2 (NM_(—)003583), DYRK3 (NM 003582), DYRK4 (NM_(—)003845) and DYRK6 (NM_(—)005734).
 45. A recombinant cell comprising at least one heterologous NFAT regulator protein or fragment or derivative thereof, and/or at least one heterologous nucleic acid encoding a NFAT regulator protein or fragment thereof.
 46. A recombinant cell overexpressing at least one mammalian NFAT regulator protein or fragment or derivative thereof.
 47. A method for identifying an agent for treating or preventing a disease or disorder associated with calcium signaling, comprising: a. assessing the effects of a test agent on an organism exhibiting the disease or disorder; and b. identifying a test agent as an agent for treating or preventing the disease or disorder if it modulates an activity, interaction, expression, or binding of at least one NFAT regulator protein or fragment or derivative thereof.
 48. The method of claim 47, wherein the disease or disorder associated with calcium signaling is rheumatoid arthritis, inflammatory bowel disease, allogeneic or xenogeneic transplantation rejection, graft-versus-host disease, aplastic anemia, psoriasis, lupus erytematosus, inflammatory disease, MS, type I diabetes, asthma, pulmonary fibrosis, scleroderma, dermatomyositis, Sjogren's syndrome, postpericardiotomy syndrome, Kawasaki disease, Hashimoto's thyroiditis, Graves' disease, myasthenia gravis, pemphigus vulgaris, autoimmune hemolytic anemia, idiopathic thrombopenia, chronic glomerulonephritis, Goodpasture's syndrome, Wegner's granulomatosis, multiple sclerosis, cystic fibrosis, chronic relapsing hepatitis, primary biliary cirrhosis, uveitis, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, Crohn's disease, ulcerative colitis, colitis/inflammatory bowel syndrome, Guilllain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, eczema, and autoimmune thyroiditis. Transplant graft rejections, acquired immunodeficiencies, common variable immunodeficiency, myocardial hypertrophy, severe combined immunodeficiency, dilated cardiomyopathy, excessive or pathological bone resorption, excessive adipocyte differentiation, obesity, or reactivation of latent viruses. 